Development of bivalent triarylalkene- and cyclofenil-derived dual estrogen receptor antagonists and downregulators

Article abstract of DOI:10.1016/j.ejmech.2020.112191

Up to 80% of mammary carcinoma initially exhibit estrogen-dependent growth, which can be treated by aromatase inhibitors or SERMs/SERDs. To increase the options after failure of the hormonal therapy with these drugs, the search for alternatives with a dif

Full text of DOI:10.1016/j.ejmech.2020.112191

European Journal of Medicinal Chemistry 192 (2020) 112191
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Development of bivalent triarylalkene- and cyclofenil-derived dual
estrogen receptor antagonists and downregulators
,
,
,
Alexandra Knox a 1, Christina Kalchschmid a 1, Daniela Schuster a b, Francesca Gaggia a,
Claudia Manzl c, Daniel Baecker a, Ronald Gust a
,
*
a Department of Pharmaceutical Chemistry, Institute of Pharmacy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, CCB e Centrum for
Chemistry and Biomedicine, Innsbruck, Austria
b Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Paracelsus Medical University, Salzburg, Austria
c Institute for Pathology, Neuropathology and Molecular Pathology, Medical University Innsbruck, Innsbruck, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
Up to 80% of mammary carcinoma initially exhibit estrogen-dependent growth, which can be treated by
aromatase inhibitors or SERMs/SERDs. To increase the options after failure of the hormonal therapy with
these drugs, the search for alternatives with a different mode of action to prevent estrogen action is of
high relevance. Therefore, this study focused on the inhibition of coactivator recruitment at the estrogen
receptor (ER) by targeted attachment of bivalent compounds at the coactivator binding site besides the
primary binding at the ligand binding domain. Eight homodimeric 4-[1-(4-hydroxyphenyl)-2-phenyl-1-
butenyl]cinnamic acid (GW7604)- or cyclofenilacrylic acid-based ER ligands with diaminoalkane linkers
(C2eC5) were synthesized and their effects on the ER subtypes were assessed in vitro. All compounds
Received 9 December 2019
Received in revised form
24 February 2020
Accepted 25 February 2020
Available online 28 February 2020
Keywords:
Estrogen receptor
Dual binding mode
Homodimeric ligands
SERDs
possessed full antagonistic potency at ERa/b as determined in a transactivation assay. Furthermore, they
exerted medium downregulatory effects dependent on the spacer length and did not stimulate the ER
expression as observed for 4-hydroxytamoxifen. The cyclofenil-derived dimer with C4 spacer (15b)
Coactivator binding site
showed the highest binding affinity to ER
7 cells with an efficiency of 38% at 1 M.
a
(RBA ¼ 79.2%) and downregulated the ER content in MCF-
m
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
as a target for selective ER modulators (SERMs) such as tamoxifen
and its active metabolite 4-hydroxytamoxifen (4-OHT, Fig. 1) [1,2].
A new approach to inhibit ER activity based on small molecules
interacting with the coactivator binding site (CABS) became more
important in medical chemistry in the last years [3,4]. The main
challenge of this strategy, however, is the relatively low binding
affinity of such compounds to the CABS owing to its size and the
low number of relevant anchors [5].
Regulation of estrogen receptor (ER) activity upon drug binding
can be achieved via different ways. So far, the ligand binding site
(LBS) within the ligand binding domain (LBD) is predominantly
chosen as target for ER agonists or antagonists. The LBS also serves
Another strategy inactivating ER-mediated signal transduction
was the bridging of ER dimers with bivalent compounds as a
consequence of simultaneous attachment at both LBSs [6e8]. This
binding mode should stabilize the dimer and effectively prevent
the formation of the activation function 2 (AF2).
In a collaboration network [9,10], we designed homodimers of
LBS binders (raloxifen, diethylstilbestrol and 4-OHT, respectively)
with spacers of various lengths. The highest binding affinity to the
ER was determined for bivalent 4-OHT compounds with spacers of
approximately 22e28 Å length, allowing the proposed attachment.
Abbreviations: 4-OHT, 4-hydroxytamoxifen; AF2, activation function 2; CABS,
coactivator binding site; DIPEA, N,N-diisopropylethylamine; DMEM, Dulbecco’s
modified eagle medium; ER, estrogen receptor; ERE, estrogen response element;
E2, estradiol; FBS, fetal bovine serum; GST, glutathione S-transferase; H, helix;
HRMS, high resolution mass spectrometry; LBD, ligand binding domain; LBS, ligand
binding site; PBS, phosphate-buffered saline; PyBOP, benzotriazol-1-yl-oxy-
tripyrrolidinophosphonium hexafluorophosphate; PGC1, peroxisome proliferator-
activated receptor gamma coactivator 1; RBA, relative binding affinity; rt, room
temperature; SERD, selective ER degrader; PA-SERD, pure antiestrogen and selec-
tive ER degrader; SERM, selective ER modulator; TFA, trifluoroacetic acid; TR-FRET,
time-resolved fluorescence resonance energy transfer.
* Corresponding author.
Interestingly, also the derivative with
a spacer length of
E-mail address: ronald.gust@uibk.ac.at (R. Gust).
These authors contributed equally to this work.
1
https://doi.org/10.1016/j.ejmech.2020.112191
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

2
A. Knox et al. / European Journal of Medicinal Chemistry 192 (2020) 112191
Fig. 1. Selective ER modulators (SERMs): tamoxifen and its active metabolite 4-OHT; selective ER degraders (SERDs): fulvestrant and ICI 164,384; SERM-SERDs: etacstil and its active
metabolite GW7604, GDC-0810, AZD-9496, LSZ102, elacestrant (RAD1901); pure antiestrogen-SERD (PA-SERD): OP-1074.
approximately 14 Å showed high ER binding. Because the com-
pound cannot simultaneously reach both LBSs of the ER dimer, it
was assumed that it binds intramolecularly within an ER monomer
with one drug moiety at the LBS and the other at the surface of the
receptor. Based on theoretical studies, the interaction within the
CABS as part of the binding mode was postulated. However, specific
interactions were predicted. Nevertheless, this study provides a
new basis for the design of bivalent drugs.
For a long time, hydrophobic side chains were utilized to design
estradiol (E2)-based SERDs (e.g. fulvestrant and ICI 164,384 [13,14],
Fig.1). Actually, most of the compounds used in clinical trials for the
treatment of hormone-dependent breast cancer bear an acrylic acid
moiety as their essential pharmacophore (e.g. GDC-0810 [15], AZD-
9496 [16], and LSZ102 [17], Fig. 1). Etacstil (4-[1,2-diphenyl-1-
butenyl]cinnamic acid, Fig. 1) [18] was the first acrylic acid deriv-
ative, which functions as an orally active tamoxifen-like SERM with
the ability to cause degradation of the ER in hormone-dependent
cells. Therefore, it was assigned as SERM-SERD.
Evidence for the suitability of the CABS as a drug target, espe-
cially regarding the binding of non-peptidic small molecules, is
offered by the crystal structure 2FSZ of the ER
b
LBD co-crystallized
Etacstil and its metabolite GW7604 [19,20] (Fig. 1) bind to ER
a
with two 4-OHT molecules. One molecule of 4-OHT is bound to the
LBS and the other one to the hydrophobic surface of the CABS [11].
These findings suggest that 4-OHT derivatives can generally be
attached to two sites within the ER.
The crystal structure 2FSZ further allows the assumption that
the unsubstituted phenyl ring deeply buried into a hydrophobic
cave caused the strongest interaction of 4-OHT at the CABS [11,12].
This part of the receptor is also important for the pharmacological
profile of fulvestrant. After the binding of the steroidal core to the
LBS, its terminal side chain is attached to that binding groove,
leading to a destabilization and consequently to a degradation of
the ER in hormone-dependent cells [13]. Drugs with such a mode of
action are representatives of selective ER degraders (SERDs).
similar to 4-OHT. The acryl side chain is located in the -channel
b
and the carboxyl group is H-bound to Asp351 inducing a confor-
mational shift of helix 12 (H12) to the CABS. Its location is slightly
different from the one caused by 4-OHT and therefore expands the
exposed hydrophobic surface of H12. The latter is made responsible
for the destabilization of ER
remains [18].
a in MCF-7 cells. The SERM activity still
Fanning et al. identified OP-1074 as another interesting com-
pound (Fig. 1). They declared it as a pure antiestrogen and selective
ER degrader (PA-SERD), which was active in tamoxifen-resistant
xenograft models [21].
Inspired by the above mentioned findings, we decided to study
the consequences of simultaneously addressing the LBS and the

A. Knox et al. / European Journal of Medicinal Chemistry 192 (2020) 112191
3
CABS on receptor binding and regulatory processes. Therefore, the
intramolecular targeting at one ER monomer instead of intermo-
lecular binding performed previously was the aim.
(Fig. 2A). This amino acid has previously been described as crucial
for binding small molecules to the CABS [3]. The C5 derivative 16a
(Fig. 3) also shows an H-bond with the charge clamp residue Lys314
(Fig. 2C), while further elongation of the aliphatic chain disturbs the
accommodation of the terminal scaffold at the CABS.
GW7604 as active metabolite of etacstil was chosen as lead
structure. Compared to 4-OHT, it shows no uterotropic (agonistic)
activity and is able to decrease ER levels as mentioned above.
Furthermore, the triarylalkene core allows the binding to both, the
LBD and the CABS, analogously to the two 4-OHT molecules offered
by the crystal structure 2FSZ. The two molecules can easily be
connected via amide bonding using various diaminoalkane spacers
(13a-16a). This design allows the adjustment of the optimal dis-
tance to achieve intramolecular binding.
A disadvantage of GW7604 is the E/Z-isomerization at its stil-
bene core. The use of (E/Z)-GW7604 results in a mixture of three
isomers (EE, EZ, and ZZ). The formation of isomers can be avoided, if
a (cyclohexylidenemethylene)dibenzene core, well known from the
SERM cyclofenil, is used [22,23]. Therefore, two molecules of the
cyclofenilacrylic acid [22] were also connected to bivalent com-
pounds (13be16b).
These theoretical investigations indicate that two different sub-
pockets at the CABS could be utilized by bivalent drugs. One cavity
is located 9e10 Å away from the nitrogen of the GW7604-amide
bound to the LBS and is addressed mainly by hydrophobic con-
tacts with residues of helices H3eH5. The second, clearly more
hydrophilic binding surface, is located 18e20 Å from the LBS.
Gln327 and charge clamp residue Lys314 constitute anchoring
points for hydrophilic residues. An intermolecular binding mode is
rather unlikely, as it would require a linker length of 22 Å [10].
The C2 spacer of the cyclofenil-based homodimer 13b (Fig. 3)
seems to be too short to reach the hydrophobic groove or mediate
relevant H-bonding to the CABS. However, 13b could adapt an
agonistic binding mode as found for an E2 derivative in the crystal
structure 2YAT [25]. It describes the ERa LBD co-crystallized with
To study the relevance of the CABS binding on the biological
activity, the 1,2-diaminoethane spacer was only bound to one
GW7604 molecule and the resulting monomer (18) was included in
this study, too. Similar derivatives of ER antagonists with an
acrylamide side chain have been investigated recently [23].
The impact of GW7604- and cyclofenil-based homodimer
binding to the LBD on the pharmacological profile was assessed
in vitro and the dependence of the activity on the employed spacer
length was elucidated.
E2 that is linked to a metal chelate. The E2 moiety is bound to the
LBS and the side chain (metal chelate) protrudes from the LBS to-
wards H7 and not H12 and therefore the agonistic potency is
retained. In our theoretical studies, only 13b was able to bind in this
manner.
The amino acid Gln327 located in the second binding surface
seems to contribute significantly to the interactions of the
cyclofenil-based homodimers at the CABS. Unlike the GW7604-
based dimers, this region is not reached with a C3 (14b, Fig. 3),
but only with a C5 spacer (16b, Fig. 3) that allows the formation of
an H-bond from the terminal cyclofenil to Gln327, being close
enough to charge clamp residue Lys314 (Fig. 2D). Additionally,
hydrophobic contacts within the lipophilic surface are possible as
well.
2. Results and discussion
2.1. Docking studies
Two molecules of GW7604 or its cyclofenilacrylic acid derivative
were connected via amide formation with diaminoalkane spacers
of various lengths. These spacers mediated a sufficient hydropho-
bicity for the interaction with the coactivator binding area and
allowed high flexibility for the attachment at both binding sites.
To estimate the optimum distance of the terminal drugs to
achieve high binding affinity, theoretical studies were performed
(for details see Experimental section). Chains ranging from C2 to C8
were chosen for both series and docking results were compared.
Due to the known binding properties of 4-OHT within the CABS, the
2.2. Chemistry
Based on the above-mentioned theoretical studies, two mole-
cules of GW7604 or the cyclofenilacrylic acid were linked by C2 to
C5 spacers, respectively. For the synthesis of the precursors, a
procedure frequently used for stilbene derivatives was used, which
has partly been described for GW7604 as well (Scheme 1) [26e29].
The synthesis is comprised of a Friedel-Crafts acylation of acyl
chloride 7a with anisole (method a) followed by a Grignard
coupling with Mg/4-bromobenzaldehyde diethyl acetal and a
dehydration step (method b), which led to a mixture of E/Z isomers
[27]. The stereo-selective Wittig-Horner reaction enabled the
introduction of an isomerically pure E-acrylate side chain (method
c; compound 10a). Ester hydrolysis (method d) and ether cleavage
(method e) yielded 4-[(E/Z)-1-(4-hydroxyphenyl)-2-phenyl-1-
butenyl]cinnamic acid (GW7604 ¼ 12a). The cyclofenilacrylic acid
12b was analogously synthesized (Scheme 1, methods a-e).
Subsequently, two acrylic acid moieties (12a or 12b) were
connected via diamide formation (method f). The phosphonium
salt based coupling reagent PyBOP [30] was chosen for this reac-
tion, having the advantage of combining high yields with an easy
handling. The dimerization to compounds 13a,b-16a,b is depended
on the temperature, the solubility of the final product, and the
applied chain length.
crystal structure 2FSZ of ER
conserved in their LBD. The ligand binding sites differ only in two
amino acids: Leu384 and Met421 in ER are replaced by Met336
and Ile373 in ER , respectively [12,24].
b was used. ERa and ERb are highly
a
b
It is noteworthy that the following theoretical considerations
are discussed based on the EE isomer, because in this case the two
biologically relevant (E)-GW7604 molecules are connected.
Furthermore, calculations with the respective EZ and ZZ isomers
pointed to an inferior binding to the LBS and CABS.
After the attachment to the LBS, the terminal drug cannot adapt
a pose comparable to 4-OHT at the CABS. In each case, it docks in a
flipped orientation with the acrylate moiety, which is directed to
the LBS, and the ethyl group which is located outside the receptor
binding pocket (Fig. 2A and B). Nevertheless, various cavities at the
CABS can be targeted.
When employing a C2 spacer, the phenyl ring of 13a (formula,
Fig. 3) is partially buried into the same groove at the CABS as 4-OHT
in 2FSZ, while the spacer is surrounded by Leu306, Met309, Ile310,
Leu331, and Trp335 forming exclusively lipophilic contacts, com-
parable to the side chain of ICI 164,384. Elongation of the spacer
leads to a more relaxed conformation: compound 14a (C3 spacer,
Fig. 3) forms an additional H-bond from the phenolic OH to Gln327
Finally, the GW7604 derivative 18, having only an 1,2-
diaminoethane side chain, was synthesized upon amide coupling
of 12a with N-Boc-1,2-diaminoethane (/ compound 17) followed
by Boc cleavage with TFA and an isolation as trifluoroacetate salt
(18, Scheme 2).
All compounds were characterized by 1H and 13C NMR spec-
troscopy as well as high resolution mass spectrometry (HRMS) and

4
A. Knox et al. / European Journal of Medicinal Chemistry 192 (2020) 112191
Fig. 2. ER
b
LBD of 2FSZ co-crystallized with two 4-OHT molecules (depicted in grey) and docked ligands (depicted in rose). Hydrophobic protein-ligand contacts are indicated by
yellow spheres, H-bonds by red and green arrows. (A) Compound 14a: The moiety occupying the LBS forms H-bonds with Arg346, Glu305, and a water molecule in a classic manner
and in this case additionally with Leu339. In the CABS an H-bond to Gln327 is formed. The receptor binding pocket with homodimer 14a (B) and 16a (C): Hydrophobic contacts are
indicated in yellow, hydrophilic interactions in blue. (D) Docked cyclofenil-based homodimer 16b: Charge clamp residue Lys314 is shown in the foreground; Gln327 forms an H-
bond with the phenolic group of the cyclofenil derivative occluding the CABS. (For interpretation of the references to color in this figure legend, the reader is referred to the Web
version of this article.)
Fig. 3. GW7604- and cyclofenil-based homodimers 13a,b, 14a,b, and 16a,b.
HPLC analyses. The purity was >95% in each case. However, the
compounds 13a-16a are a mixture of isomers (EE, EZ, ZZ), because
GW7604 used in the synthesis was not isomerically pure (see
above). The NMR spectra showed signals, which were assigned to
the isomers by 2D NMR (see Supplementary data).
Using the example of 14a, an isomer ratio of EE:EZ:ZZ ¼
25:50:25 was found and remained constant throughout three days
of incubation in MeOH/2x PBS at 37 ꢀC (see Experimental section
and Supplementary data), similar to the findings on 4-OHT-derived
homodimers reported by Shan et al. [10].
Isomerization of a 4-hydroxystilbene or triarylalkene core dur-
ing the synthesis is a general problem in this class of compounds
[31,32]. Classical synthesis routes leading to GW7604 other than
that used in this study, e.g. McMurry and Heck-coupling reactions
[22], cannot improve the E/Z ratio and were found to have less total
yield as well as difficulties in scaling up.
However, it should be mentioned that it is possible to separate
(E)-GW7604 from the mixture by crystallization, but geometric
isomerism occurs immediately in solution as already determined
for 4-OHT. (Z)-4-OHT undergoes 20e30% isomerism after

A. Knox et al. / European Journal of Medicinal Chemistry 192 (2020) 112191
5
Scheme 1. Synthesis pathway of compounds 12a,b and 13a,b-16a,b: Reagents and conditions: (a) anisole, AlCl3, anh. DCM, 0 ꢀC to room temperature (rt), 2.5 h (yields: 61e76%); (b)
i) Mg, 4-bromobenzaldehyde diethyl acetal, anh. THF, rt, 4 h; ii) EtOH, HCl conc., reflux, 5 h (yields: 63e73%); (c) trimethyl-/triethylphosphonoacetate, potassium bis(trimethylsilyl)
amide, anh. THF, 0 to ꢁ78 ꢀC to rt, 20 h (yields: 76e85%); (d) THF:EtOH ¼ 1:1, 2N KOH, rt, 24 h (yields: 85e99%); (e) BBr3, anh. DCM, 0 ꢀC, 2 h (yields: 66e92%); (f) DIPEA, PyBOP,
anh. DMF, anh. DCM, 0 ꢀC to rt to 45 ꢀC, 20 h to 3 d (yields: 28e75%).
incubation for two days in cell-free culture medium at 37 ꢀC and
also in ethanol stock solutions at ꢁ20 ꢀC [32,33]. Experiments that
lasted up to six months showed an equilibrium of E:Z ¼ 50:50,
regardless of the applied conditions [34].
GW7604 is mostly investigated as an E/Z mixture [23,35], but it
was reported that the effects at the isolated ER [22] as well as in
cell-based assays can be assigned to the more active E isomer [35].
It is noteworthy that the bivalent cyclofenil derivatives are iso-
merically pure.
(RBA) compared to E2 (100%), were assessed in a TR-FRET assay on
the isolated human ER /ER LBDs. 4-OHT, GW7604, and fulvestrant
were used as references.
4-OHT showed an RBA value of 14.7% for ER
The exchange of the basic side chain with an acrylic acid moiety
(GW7604) reduced the affinity to 6.2% (ER ) and 27.1% (ER ). Both
compounds were 4.5-fold more selective for ER (Table 1).
Derivatization of GW7604 with an 1,2-diaminoethane chain (/
18) reduced the binding to ER
further (RBA ¼ 2.6%), while that to
ER
remained unchanged (RBA ¼ 23.9%). Interestingly, an addi-
tional GW7604 moiety at 18 resulting in the bivalent compound 13a
did not influence the binding to ER
(RBA ¼ 2.2%), but strongly
reduced the interaction with ER
(RBA ¼ 1.5%).
The elongation of the spacer by one (/ 14a) or three methylene
groups (/ 16a) increased the RBA to ER (25.9% or 9.4%,
a
b
a
and 60.7% for ERb.
a
b
b
a
b
2.3. Biological evaluation
a
b
2.3.1. Ligand binding affinity
The binding affinities of the GW7604- or cyclofenil-based
homodimers to the LBS, expressed as relative binding affinity
a

6
A. Knox et al. / European Journal of Medicinal Chemistry 192 (2020) 112191
Scheme 2. Synthesis of monomer 18.
Reagents: (a) DIPEA, PyBOP, anh. DMF, anh. DCM, rt, 24
h (yield: 51%); (b)TFA, anh. DCM, 0 ꢀC, 2 h, (quant.).
For the interpretation of the results concerning ERa, the theo-
Table 1
In vitro competitive binding assay.
retical studies are helpful, because the TR-FRET assay studied the
drug-ER interactions on the molecular level. The derivation of
GW7604 (/ 18) slightly reduced the affinity to the ER. Even the
binding of a second GW7604 molecule to 18 (/ 13a) did not
influenced the RBA. The spacer is too short to reach the hydro-
phobic pocket at the CABS located at a distance of 9e10 Å of the LBS.
This is possible if the spacer is elongated by one methylene group
(C3 spacer, / 14a). Docking analyses documented further that the
optimum spacer length (C5 spacer, / 16a) allowed H-bonding of
the second GW7604 moiety of the dimer with Gln327 and Lys314,
respectively, approximately 18e20 Å away from the LBS.
The RBA values of the compounds 13be16b were higher than
that of the respective GW7604 dimers, which very likely results
from the presence of the isomeric mixture in case of GW7604 di-
mers. The molecular docking studies revealed that the EE isomers,
which constitute only 25% of the GW7604 series, possess the spatial
structure necessary for the simultaneous binding to the LBS and
CABS. The EZ (50%) and ZZ (25%) isomers contribute to this effect to
a much smaller extent, because of an unfavorable attachment to the
LBS and/or the CABS (see molecular docking).
Compound
TR-FRETa ER
IC50 [nM]
a
RBA ER
%
a
TR-FRETa ER
IC50 [nM]
b
RBA ER
b
%
13a
14a
15a
16a
13b
14b
15b
16b
15.1
1.28
18.9
3.52
6.43
1.67
0.42
2.35
13.6
2.24
5.35
2.85
0.33
5.6
0.51
4.0
2.2
25.9
1.9
9.4
5.1
19.7
79.2
14.1
2.6
14.7
6.2
67.7
22.2
44.0
15.5
27.5
34.0
15.7
17.5
4.26
1.68
3.77
10.2
1.02
8.3
8.8
1.5
4.6
2.3
6.6
3.7
3.0
6.5
5.8
23.9
60.7
27.1
10.0
100
17.5
9.1
11.9
19.8
9.5
0.95
2.64
0.59
0.10
0.76
2.8
0.89
1.39
0.83
0.19
2.7
18
0.66
1.00
1.06
2.5
4-OHT
GW7604
fulvestrant
E2
11.6
100
0.50
a
Displacement of fluorescent-labeled E2 from ER
a or ERb by GW7604- or
cyclofenil-based homodimers. IC50 values represent means SD of ꢂ 3 independent
experiments.
respectively). The C4 derivative 15a (RBA ¼ 1.9%) showed the same
affinity as 13a. It should be mentioned that the RBAvalue of 16a was
comparable to that of fulvestrant (RBA ¼ 11.6%) and the binding
affinity of 14a was even higher than that of 4-OHT (RBA ¼ 14.7%).
Interestingly, GW7604-derived compounds showed higher af-
2.3.2. Coactivator recruitment
In a further TR-FRET experiment, the influence of the com-
pounds on the attachment of the coactivator PGC1 at the ERa LBD
finity to ER
FRET increased in the series 15a (0.83) < 16a (1.42) ¼ 13a
(1.47) < 14a (5.63) (compare RBAs at ER and ER in Table 1).
In the cyclofenil-based series all compounds possessed equal
than GW7604
a compared to ERb. The ERa/ERb ratio based on the TR-
was investigated. E2 showed a recruitment of 100% at 10 nM (for
details see Supplementary data). The bivalent compounds did not
induce PGC1 binding up to a concentration of 1 mM. Interestingly,
the cyclofenil derivative 13b was also inactive, although an
agonistic binding mode is generally possible according to theoret-
ical studies (see above).
The coactivator binding was also investigated in the presence of
E2 (at EC50 ¼ 4 nM) by taking the examples of GW7604 as well as
14a,b and 15a,b.
a
b
(13b, RBA ¼ 5.1%) or higher affinity to ER
a
(RBA ¼ 6.2%). An extraordinary high RBA value of 79.2% was
determined for 15b (C4 derivative). This finding contradicts the
molecular docking studies that proposed a stable binding pose at
the CABS only for the C5 derivative 16b (RBA ¼ 14.1%). Compound
14b (C3 spacer, RBA ¼ 19.7%) showed a 3-fold higher affinity than
GW7604, too.
GW7604 completely antagonized the E2 stimulating effects after
an incubation time of 10 min only at concentrations higher than
It is noteworthy that also the cyclofenil derivatives demon-
strated subtype selectivity for ER
FRET)): 13b (1.38) < 16b (2.43) < 14b (6.57) < 15b (12.18).
1
m
M. After incubation for 30 min, a maximum of about 50% of
inhibition was observed at 5 M.
The bivalent compounds were distinctly more active. All
a (ERa/ERb ratio (based on the TR-
m

A. Knox et al. / European Journal of Medicinal Chemistry 192 (2020) 112191
7
compounds inhibited the coactivator recruitment, which is induced
by E2 more than 75% at the lowest concentration of 5 nM and
antagonized the E2 effect completely (100%) at 100 nM (Fig. 4). In
contrast to GW7604, 14a,b and 15a,b were still active at 100 nM
(about 50% inhibition) after an incubation for 30 min. At 1 mM, 14a
and 15a even completely prevented coactivator binding.
These findings clearly indicate that contrary to GW7604 the E2-
induced coactivator recruitment can be effectively blocked by
bivalent compounds.
extraordinarily high ER downregulation (see below). The calcula-
tion of its IC50 values was not feasible.
2.3.4. Estrogen receptor downregulation
Next, the influence of the compounds on the ER
MCF-7 cells was studied. The ER expression after 24 h of incuba-
tion with the respective compound (1 M) was analyzed by
Western blotting. -actin was used as loading control (Fig. 6).
a content in
a
m
b
Additionally, the receptor protein was quantified by In-Cell West-
ern analyses (Table 3).
2.3.3. Inhibition of transactivation
The treatment of the MCF-7 cells with the SERD fulvestrant for
To obtain further information about the influence of the com-
pounds on the signal transduction, a cellular assay with U2OS cells
24 h led to an almost complete degradation of ERa. Tamoxifen and
especially its active metabolite 4-OHT are SERMs with a mixed
agonistic/antagonistic pharmacological profile and stabilize the
receptor [36,37]. Therefore, 4-OHT strongly increased the protein
level, as visible in the Western blot (Fig. 6).
The comparison of the blots obtained for 4-OHT and GW7604
demonstrates the relevance of the side chain for interference with
transiently transfected with the ER plasmids pSG5-ER
a or pSG5-
ER
b
and the reporter plasmid p(ERE)2-lucþ as well as pRenilla-
CMV for standardization was used. The expression of luciferase is
a measure for binding ER dimers to the estrogen response elements
(EREs) at the reporter plasmid.
None of the compounds caused agonistic effects at 0.1
m
M or
ER
chain), GW7604, which has an acrylic acid moiety, caused slight
degradation. The GW7604 derivative 18 increased the ER content
in the cells. The stimulating properties depended on the presence
of a free, under physiological conditions cationic side chain. The
binding of a second GW7604 molecule to 18 resulted in the un-
charged bivalent compound 13a with low degradation potency. The
latter was increased by an elongation of the spacer (/ 14a-16a).
The same trend was observed for the cyclofenil derivatives
13be16b.
a expression. In contrast to 4-OHT (dimethylaminoethanol side
1.0
m
M, which confirmed the inability of coactivator recruitment
(see above). Alternatively, this could also be caused by the blocking
of ER dimerization upon drug binding, preventing the interaction
with the reporter plasmid, too.
a
In contrast, all bivalent compounds proved to be potent antag-
onists and inhibited the transactivation of E2 at ER
a (E2 conc.:
0.03 nM) and ER
concentration-ER
b
a
(E2 conc.: 0.3 nM), respectively. The resulting
activation curves are depicted in Fig. 5 and the
IC50 values are listed in Table 2.
Interestingly, the most potent GW7604 derivative was 18 with
Based on the Western blot analyses, the bivalent compounds
an IC50 ¼ 5.33 nM at ER
gene activation comparable to 4-OHT (ER
IC50 ¼ 0.99 nM). Derivation of 18 to the GW7604 derivatives 13a-
16a reduced the antagonistic effects at ER
a
and IC50 ¼ 3.62 nM at ER
b
. It prevented
induced ERa degradation without carrying an acrylic acid side
chain. Therefore, it was of interest to quantify the cytosolic receptor
in an In-Cell Western immunoassay (Table 3).
a
: IC50 ¼ 2.28 nM; ER
b:
a
(IC50 ¼ 136e290 nM).
As expected, at a concentration of 1
mM only 4-OHT and 18
Compounds 14a (IC50 ¼ 136 nM) and 16a (IC50 ¼ 160 nM) were still
2-fold more active than GW7604 (IC50 ¼ 238 nM). Compounds 13a
(IC50 ¼ 260 nM) and 15a (IC50 ¼ 290 nM) were less effective, but
upregulated the ER protein level compared to the DMSO control
a
(100%) to 263% and 165%, respectively. The efficacy of 13a was low
(11%), but was enhanced by elongation of the spacer length. 14a
caused the highest ER downregulation to 51% (efficacy: 49%)
compared to the control, 15a (efficacy: 32%) and 16a (efficacy: 36%)
were slightly less active. The same trend was observed in the
cyclofenil-based series. The most active compound was 14b (effi-
cacy: 51%) followed by 15b (efficacy: 38%), 16b (efficacy: 21%), and
13b (efficacy: 8%).
Compared to fulvestrant (efficacy: 100%), the compounds were
SERDs of moderate potency (Fig. 7), whereby only 14a and 14b
reached downregulation of 50% and were basically as efficient as
GW7604 (efficacy: 56%).
reached the GW7604 potency. The inhibitory effects at ERb were
comparable to that at ER
possessed slight ER subtype selectivity.
Within the cyclofenil series, except for compound 14b (ER
a
(Table 2). Only 13a (IC50 ¼ 78.7 nM)
b
a:
IC50 ¼ 565 nM; ER : IC50 ¼ 304 nM), all compounds were more
b
potent antagonists than their GW7604 analogues. The most effi-
cient inhibitors were 15b and 16b with IC50 ¼ 88.3 and 73.0 nM at
ER
a
and IC50 ¼ 54.8 and 49.4 nM at ER
b, respectively.
It is worth mentioning that fulvestrant caused an unusual con-
centration activity curve, completely inhibiting the E2-stimulated
luciferase expression even at the lowest concentration (0.05 nM).
Such an activity profile can be explained by the induction of an
In conclusion, cationic compounds such as 4-OHT or 18 showed
SERM-like activity. Although they antagonized the E2 effects in the
Fig. 4. Inhibition of coactivator recruitment by concomitant administration of bivalent compounds 14a,b and 15a,b with E2 (4 nM). Incubation time: 10 min (left) and 30 min
(right).

8
A. Knox et al. / European Journal of Medicinal Chemistry 192 (2020) 112191
Fig. 5. Inhibition of ERa transactivation in U2OS cells transiently transfected with the ER plasmids pSG5-ERa
and p(ERE)2-lucþ. GW7604-based homodimers (left) and cyclofenil-
based homodimers (right).
Table 2
Luciferase reporter gene assay (ER
a
and ER
b
) using U2OS cells, transiently trans-
fected with plasmids pSG5-ER
a
or pSG5-ER
b
and the reporter plasmid p(ERE)2-lucþ
.
Compound
Transactivation ER
IC50 [nM]
a
Transactivation ER
IC50 [nM]
b
a
a
13a
14a
15a
16a
13b
14b
15b
16b
260
136
290
160
131
565
88.3
73.0
5.33
2.28
238
n.d.b
70
28
57
52
14
43
2.3
6.4
3.23
1.34
74
78.7
153
215
256
216
304
54.8
49.4
3.62
0.99
154
n.d.b
9.4
53
155
112
135
160
28.8
33.1
1.43
1.25
53
Fig. 7. Percent efficacy related to fulvestrant. Compound 18 and 4-OHT showed
upregulation. Values represent means þ SD of ꢂ3 independent experiments.
18
4-OHT
GW7604
fulvestrant
interaction with the target molecule, the ER.
a
IC50 values represent means SD of ꢂ3 independent experiments.
n.d.: not defined.
b
Generally, the reduced intracellular ERa content can be seen as
the consequence of a lower expression or an increased degradation
of the receptor protein. Therefore, the influence of the ubiquitin-
proteasome pathway [38] on the degradation was studied on the
examples 14a, GW7604 and fulvestrant. The cells were incubated
with the respective compound (1
proteasome inhibitor MG-132 (1
m
M) either with or without the
m
M) [39]. The receptor protein
content was analyzed by Western blotting.
As depicted in Fig. 8, MG-132 marginally blocked the effects of
fulvestrant and GW7604. The degradation caused by 14a was un-
Fig. 6. ER
(1 mM) for 24 h.
a expression levels in MCF-7 cells after incubation with the compounds
affected by MG-132, which indicates that the reduced ER
is not caused by the ubiquitin-proteasome pathway.
a content
luciferase reporter gene assay (Table 2), they stimulated ER
a
It is well known that the ER enters the proteolytic pathways
through alternate mechanisms depending on the ligand-induced
structure modifications. In the case of fulvestrant and GW7604,
expression in MCF-7 cells. In contrast, GW7604 and its dimers as
well as the cyclofenil analogues were SERDs of medium potency.
The most active compounds 14a and 14b showed high binding af-
distinct effects on the compartmentalization of the ERa within the
finity to ER
a
and were pure antagonists in the transactivation assay
cell have been revealed. The E2-induced receptor shape enables the
recognition of the proteasome and the subsequent degradation.
and can be assigned to the group of PA-SERDs. The biological ac-
tivity depended on the spacer length, which indicates a selective
Table 3
ERa levels in MCF-7 cells determined by In-Cell Western immunoassay.
Compound
% ER
a
Remaininga
% Efficacyb
Compound
% ER
a
Remaininga
% Efficacyb
13a
14a
15a
16a
18
GW7604
4-OHT
89.0 1.6
50.6 4.6
68.0 5.1
63.9 3.1
165.0 4.0
44.3 12.1
263.0 9.0
11
49
32
13b
14b
15b
16b
fulvestrant
DMSO
91.8 5.6
49.1 6.7
62.0 7.4
78.5 9.2
0
8
51
38
21
100
0
36
c
56
100
c
a
ER
a
levels compared to solvent control (DMSO). Values represent the mean of ꢂ3 independent experiments SD.
Efficacy at 1 M, calculated as downregulation of ER compared to the efficacy of reference compound fulvestrant.
Upregulation.
b
c
m
a

A. Knox et al. / European Journal of Medicinal Chemistry 192 (2020) 112191
9
Fig. 8. ERa expression levels in MCF-7 cells after 4 h of incubation with 1 mM of the
compounds, either with or without the protease inhibitor MG-132 (1 mM).
2.3.5. Solubility and cellular uptake
Essential parameters for the interpretation of cellular effects are
the water solubility and the cellular uptake of drugs.
Compounds which are part of the GW7604 series showed higher
Fig. 9. Antiproliferative effects on MCF-7 breast cancer cells, determined in the crystal
violet assay. T is the biomass of cells treated with compounds for 72 h; C is the biomass
of control cells after 72 h.
solubility in aqueous solutions (13a: >40
19 M; 16a: 34 M) than fulvestrant (11.1
rivatives are more lipophilic, which hampers the dissolution in
water (saturation concentration: 13b: 6.5 M; 14b: 15.4 M; 15b:
10 M; 16b: 10 M). In all cases, however, it was possible to reach
mM; 14a: 24
mM; 15a:
m
m
mM). Cyclofenil de-
m
m
The curve of fulvestrant is flat as well, but started with lower T/C
m
m
values (T/C ¼ 64% at 50 nM) and reached T/C ¼ 33% at 10
mM, which
the concentrations necessary for the above described cell culture
experiments.
Uptake studies (at 10 mM, 24 h of incubation) were performed on
the examples 18, 15a, and 15b, based on the inherent fluorescence
of their cinnamide scaffold.
allowed the calculation of IC50 ¼ 4.93
m
M.
All bivalent compounds showed concentration-activity curves
similar to GW7604, which does not allow a reasonable calculation
of the IC50 values. The use of higher concentrations was restricted
by the insufficient solubility above 20
mM. The reduced cell mass at
All compounds were taken up in ER-positive MCF-7 cells
(Table 4), 15a and 15b to the same amount of about 5 nmol/mg
protein. Interestingly, the positive charge of 18 enabled an
approximately 7-fold enrichment (35.3 nmol/mg protein)
compared to the other compounds. Because 15a and 18 caused
comparable intracellular amounts in MCF-7 and the fibroblast-like
COS-7 cells (about 4 and 34 nmol/mg protein, respectively), it is
very likely that the uptake is not receptor mediated. The content of
15b was even higher in COS-7 cells (11.7 nmol/mg protein). The
parent compound GW7604 did not provide adequate relative
fluorescence intensity and therefore could not be used as reference
for the cellular uptake studies.
10 M (see Table 1 in Supplementary data) generally documented
antiproliferative effects. Analogous results were achieved with the
cyclofenil derivatives, whereby lower water solubility allowed
m
compound concentrations only up to 10
Interestingly, compound 18 caused the expected sigmoid
concentration-activity curve, from which an IC50 ¼ 4.95
(Table 5) was calculated. Thereby, 18 was as active as 4-OHT
(IC50 ¼ 4.94 M). The antiproliferative effects of both compounds
were not restricted to MCF-7 cells. They also reduced the growth of
ER-negative, non-tumorous COS-7 cells (18: IC50 ¼ 5.95 M; 4-OHT:
M). Such unspecific effects are well known for 4-OHT
mM.
mM
m
m
IC50 ¼ 8.69
m
and are associated with cancerogenic DNA adducts determined for
instance in hepatocytes [42] and the endometrium [43]. In contrast,
fulvestrant was 7-fold more effective against MCF-7 cells
2.3.6. Antiproliferative effects
The influence of the bivalent drugs on the growth of hormone-
dependent, ER-positive MCF-7 breast cancer cells was investigated
in a standardized crystal violet assay. For this purpose, the cells
were incubated with compounds for 72 h and the cell mass was
quantified by staining and measuring the absorbance [40]. The
reduction of cell mass correlates with the antiproliferative effects of
the drugs.
GW7604 as a reference caused a very flat concentration-activity
curve and reached a T/C value of 75.5% at 10 mM. Therefore, it was
not feasible to calculate the IC50 value from this curve (Fig. 9). The
cellular behavior differs from that described in the literature where
IC50 values up to 2 mM are described [41]. This discrepancy might be
(IC50 ¼ 4.93
m
M) compared to COS-7 cells (IC50 ¼ 34.0
mM).
The antiproliferative effects were further evaluated against
MDA-MB-231 and SKBr-3 breast cancer cells. The bivalent de-
rivatives were inactive at the concentration used (see Supple-
mentary data). In contrast, 4-OHT as reference reduced the cell
mass at 10
m
M as well as 20
m
M independent of the cell line used. It
M to 79% (MDA-MB-231) and 58%
M even cytocidal effects were
inhibited the proliferation at 10
(SKBr-3), respectively. At 20
observed (MDA-MB-231: T/C ¼ ꢁ80%; SKBr-3: T/C ¼ ꢁ81%). These
findings confirmed the ER-independent influence of 4-OHT on the
cell growth. They are further an indication that the bivalent com-
pounds exert their biological activity mainly due to the interaction
m
m
the consequence of different conditions used in the assays, e.g. the
number of cells seeded and the incubation time. For instance, Fan
et al. determined a greater distance between test and control curves
for GW7604 only with increased incubation time. After 72 h the
differences of antiproliferative effects were marginal [27].
with ER
while SKBr-3 are negative concerning both subtypes (ER
ER -negative).
a
. MDA-MB-231 cells are ER
a
-negative and ER
b
-positive,
a
-negative,
b
Table 5
Table 4
Antiproliferative effects against ER-positive MCF-7 and the fibroblast-like COS-
7 cells.
Cellular uptake of compounds 15a, 15b, and 18 (10
m
M) after 24 h of incubation.
Compound
MCF-7 cells [nmol
compound/mg
protein]a
COS-7 cells [nmol
compound/mg
protein]a
Compound
MCF-7
COS-7
IC50
[m
M]a
IC50 [m
M]a
15a
15b
18
4.67
5.22
35.3
1.28
1.68
7.0
3.92
11.7
33.7
0.49
0.8
7.8
18
4.95
4.94
4.93
1.43
0.26
1.06
5.95
8.69
34.0
1.03
1.20
4.1
4-OHT
fulvestrant
a
a
Values represent means SD of ꢂ3 independent experiments.
Values represent means SD of ꢂ3 independent experiments.

10
A. Knox et al. / European Journal of Medicinal Chemistry 192 (2020) 112191
2.3.7. Antimetabolic activity
agonistic side effects. In contrast to 4-OHT, they reduced the ERa
In addition to the antiproliferative effects, the antimetabolic
activity of the compounds was quantified in a modified MTT assay
(EZ4U). The mitochondrial guided transformation of a tetrazolium
salt to its colored formazan is a tracer for cell viability. The com-
pounds were tested for activity on the MCF-7 and COS-7 cell lines
(Fig. 10). Regarding their low water solubility, cyclofenilacrylic acid
derivatives 13be16b and GW7604 were tested only up to a con-
content in MCF-7 cells, which was not prevented by the protea-
some inhibitor MG-132. Therefore, they modulate the ER expres-
sion rather than the ER degradation. The cyclofenil-based
homodimers seem to be slightly superior to the GW7604-based
homodimers. However, this effect could be just obtained through
the influence by the active isomers, as previously discussed.
The conformational change caused by simultaneously blocking
both the LBS and the CABS is not clear yet, but it probably leads to
an uncontrolled orientation of H12, prevention of AF2 formation
and inhibition of coactivator binding. Introduction of a C3 spacer
between two drug molecules appears to offer the best conditions
for degradation. A complete destabilization as observed for ful-
vestrant is unlikely, because of the missing long side chain. A
fulvestrant-like ER binding might impair receptor dimerization and
energy-dependent nucleocytoplasmic shuttling, thereby blocking
nuclear localization of the receptor [46,47].
centration of 10 mM.
The results obtained from the modified MTT assay partially
differed from that of the crystal violet assay. GW7604 was
completely inactive towards MCF-7 cells at the used concentra-
tions. Among the GW7604 derivatives, 13a and 15a reduced the
metabolic activity to 45% and 70% at 20
rivatives 14b, 15b, and 16b showed a reduction to 53%, 69%, and 50%
at 10 M, respectively. Interestingly, compound 18 (60% at 20 M)
was less active than 4-OHT (18% at 20 M), but showed similar
effects to fulvestrant (61% at 20 M).
mM. The cyclofenil de-
m
m
m
m
Furthermore, the carboxylate of GW7604 is not essential for the
ER downregulation. Additional contacts in the CABS upon deriva-
tion led to the same effects. The underlying drug design concept
additionally enabled to prevent the stimulating effects observed by
4-OHT as well as by the use of cationic or uncharged side chains.
None of the homodimeric compounds had a considerable in-
fluence on the metabolic activity regarding COS-7 cells at the
employed concentrations, which indicates again selectivity to-
wards the ER-positive tumor cells.
3. Conclusion
4. Experimental
Bivalent GW7604 and cyclofenilacrylic acid derivatives were
synthesized to simultaneously target the ligand and the coactivator
binding site within one ER monomer. By attaching at two sites
within the ER LBD, the affinity to the ER should be strengthened
and provide a mode of action different from well-known SERMs
such as 4-OHT. This approach differs from the design of SERMs in
order to optimize the effects of 4-OHT or GW7604 [44,45],
respectively.
Based on the theoretical studies, it was obvious that the CABS
can be addressed using Gln327 or Lys314 as H-bonding anchors.
Other than that, hydrophobic contacts are suitable interactions at
the ER surface for ligand binding as well. This part of the ER is easily
accessible from the LBS using a C3, C4, or C5 spacer and a second
drug molecule.
4.1. Chemistry
4.1.1. Computational design of the homodimers
The 2D chemical structures were generated with ChemDraw
(version 14.0.0.117). Energy minimization of the homodimers was
performed using the MM2 force field calculation method of
ChemBio3D Ultra [48]. GOLD suite 5.2 [49] was employed for the
docking experiments. The binding site was defined in a 20 Å radius
around the oxygen of Leu306 (atom coordinates 73.479, 8.386,
22.166). Other than that, default settings were used and no con-
straints were set. Ten binding postures per ligand were chosen and
the results were examined in LigandScout 4.1 alpha4 [50]. Docking
poses were evaluated visually and the GoldScore and the ChemPLP
scoring function were compared, respectively. To ensure that there
are no major differences in the binding mode to the LBS and the
The most interesting compound, the cyclofenil derivative 15b,
displayed ER downregulatory potency of 38% at 1 mM (fulvestrant:
100%) and was able to displace E2 with an RBA ¼ 79.2% from its
binding site. It completely prevented the recruitment of the PGC1
coactivator peptide and abolished the E2-induced transactivation
CABS of ER
b
compared to those of ER
a, the two crystal structures of
ER (3ERD [51]) and ER
a
b
(3OLS [52]) were implemented and
aligned using the program PyMol [53]. Prior to docking, the
redocking of the two co-crystallized 4-OHT molecules was carried
out for both the LBS and the CABS (root-mean-square
deviation ¼ 0.530 and 0.810, respectively).
in U2OS cells, transiently transfected with the plasmids pSG5-ER
a
and the reporter plasmid p(ERE)2-lucþ with an IC50 ¼ 68 nM.
All bivalent ER inhibitors were full antagonists without having
Fig. 10. Percentage metabolic activity compared to the DMSO control (set to 100%). The means þ SD of ꢂ3 independent experiments is shown.

A. Knox et al. / European Journal of Medicinal Chemistry 192 (2020) 112191
11
4.1.2. General
(700 MHz, DMSO‑d6, EE:EZ:ZZ ¼ 25:50:25):
d
0.86 (t, 3J ¼ 7.3 Hz, 6H,
All reagents were purchased from Sigma-Aldrich, TCI, VWR, and
Alfa Aesar and were used without further purification. All solvents
were distilled before usage. Anhydrous solvents were obtained by
distillation under argon over an appropriate drying agent. Anhy-
drous reactions were performed under an inert argon atmosphere
using oven-dried glassware, septa and syringes. The reactions were
monitored using thin-layer-chromatography (TLC) on Polygram®
SIL G/UV254 silica gel polyester sheets (Macherey-Nagel, Düren,
Germany), and visualized with UV light (254 or 366 nm). Chro-
CH2CH3), 2.39 (q, 3J ¼ 6.9 Hz, 2H, CH2CH3, Z isomers), 2.43 (q,
3J ¼ 7.4 Hz, 2H, CH2CH3, E isomers), 3.22 (s, 1H, NHCH2CH2NH, EE
isomer), 3.27 (s, 2H, NHCH2CH2NH, EZ isomer), 3.32 (s, 1H,
NHCH2CH2NH, ZZ isomer), 6.42 (d, 3J ¼ 8.5 Hz, 2H, ArH, Z isomers),
6.45 (d, 3J ¼ 16.5 Hz, 1H, CHCHCONH, E isomers), 6.55e6.67 (m, 2H,
ArH þ CHCHCONH, Z isomers), 6.77 (d, 3J ¼ 8.4 Hz, 2H, ArH, E iso-
mers), 6.80e6.84 (m, 2H, ArH, E isomers), 7.00 (d, 3J ¼ 8.0 Hz, 2H,
3
ArH, E isomers), 7.03e7.25 (m, 14H, ArH), 7.26 (d, J ¼ 15.4 Hz, 1H,
CHCHCONH, E isomers), 7.41e7.50 (m, 1H, CHCHCONH, Z isomers),
7.50e7.62 (m, 2H, ArH, Z isomers), 8.06e8.29 (m, 2H, NH), 9.21 (s,
matography purification on Silica gel 60
Å was performed
employing either classic standard procedures or by using a Biotage
Isolera 1 Flash purification system. 1H and 13C NMR spectra were
obtained on a Varian Gemini-200 (now Agilent), 400 MHz Avance 4
Neo (Bruker), 600 MHz Avance II (Bruker) or 700 MHz Avance 4 Neo
(Bruker) spectrometer. Deuterated chloroform (CDCl3), dimethyl
sulfoxide (DMSO‑d6), acetone ((CD3)2CO) and methanol (CD3OD)
were used as solvents for NMR. The chemical shifts in ppm were
used as a reference to tetramethylsilane or the solvent peak.
Coupling constants (J) are listed in Hertz (Hz). The purity of final
compounds 13e16 as well as stability measurements based on
compound 14a were performed by using HPLC (Shimadzu) on a C18
1H, OH), 9.46 (s, 1H, OH). 13C NMR (176 MHz, DMSO‑d6):
d 13.33,
13.38, 28.47, 28.61, 28.99, 38.55, 38.59, 38.60, 38.64, 114.45, 115.11,
121.60, 121.91, 126.15, 126.30, 126.66, 127.46, 127.90, 127.96, 129.35,
129.37, 129.61, 130.22, 130.81, 131.40, 132.19, 133.02, 133.23, 133.76,
137.87, 138.25, 138.27, 138.34, 138.36, 140.72, 141.67, 141.77, 141.84,
144.54, 144.68, 155.43, 156.29, 165.17, 165.20, 165.23. HRMS (m/z):
calculated for C52H47N2O4 [MꢁH]-: 763.3536, found: 763.3577.
4.1.3.1.2. (E)-N,N’-(Propane-1,3-diyl)bis[3-(4-((E/Z)-1-(4-
hydroxyphenyl)-2-phenylbut-1-en-1-yl)phenyl)acrylamide]
(14a).
14a was synthesized according to the general procedure described
above: 100 mg of 12a (0.27 mmol), 147 mg of PyBOP (0.28 mmol),
0.062 mL of DIPEA (0.35 mmol) and 9.5 mg of 1,3-diaminopropane
(0.13 mmol) in 1.0 mL of anh. DMF. The mixture was stirred at rt for
48 h. 14a was obtained as a yellowish, sparkling powder (68 mg,
0.087 mmol, 63%). Purity: 98.8%. 1H NMR (600 MHz, CD3OD,
column (Knauer) employing 10
m
L
injection volume, an
acetonitrile-water gradient (flow rate 1.2 mL/min) with UV detec-
tion at 254 or 283 nm and 37 ꢀC oven temperature. All final com-
pounds were ꢂ95% pure (for HPLC spectra see Supplementary
data). High resolution mass spectra were obtained from an Orbitrap
Elite mass spectrometer (Thermo Fisher Scientific). Detailed
experimental data for the preparation and characterization of
compounds 8e12 are described in the Supplementary data.
EE:EZ:ZZ ¼ 28:47:25):
d
0.92 (t, 3J ¼ 7.4 Hz, 6H, CH2CH3), 1.74 (p,
3J ¼ 6.8 Hz, 0.6H, NHCH2CH2CH2NH, EE isomer), 1.78 (p, 3J ¼ 6.8 Hz,
0.9H, NHCH2CH2CH2NH, EZ isomer), 1.83 (p, 3J ¼ 6.7 Hz, 0.5H,
NHCH2CH2CH2NH, ZZ isomer), 2.46 (2xq,1.9H, 3J ¼ 7.3 Hz, CH2CH3, Z
isomers), 2.51 (q, 3J ¼ 7.4 Hz, 2.1H, CH2CH3, E isomers), 3.35 (q,
3J ¼ 6.9 Hz, 2H, NHCH2CH2CH2NH, EZ isomer), 3.39 (t, 3J ¼ 6.8 Hz,
4.1.3. Synthesis of the final compounds
If possible, the signals in the 1H NMR spectra were assigned to
the EE, EZ, or ZZ isomer or to their respective moieties (expressed as
E and Z isomers). The proton proportions (with one decimal place)
express the isomeric ratio. The signals in the 13C NMR spectra were
ascribed to the respective isomers based on 14a and 15a as repre-
sentatives. The preparation of GW7604 (12a) and the cyclofenil
derivative 12b was performed according to known procedures and
was further optimized (see Supplementary data).
1H, NHCH2CH2CH2NH, ZZ isomer), 6.41 (d, J ¼ 7.9 Hz, 1.9H, ArH, Z
3
isomers), 6.42e6.47 (m, 1H, CHCHCONH, E isomers), 6.59 (d,
3J ¼ 15.8 Hz, 0.5H, CHCHCONH, EZ isomer), 6.61 (d, 3J ¼ 15.8 Hz,
0.5H, CHCHCONH, ZZ isomer), 6.64e6.68 (2xd, 3J ¼ 8.6 Hz, 1.9H,
ArH, Z isomers), 6.77 (d, 3J ¼ 7.8 Hz, 2.1H, ArH, E isomers), 6.85e6.88
(2xd, 3J ¼ 8.3 Hz þ 8.4 Hz, 2.1H, ArH, E isomers), 7.03 (d, 3J ¼ 7.7 Hz,
2.1H, ArH, E isomers), 7.08e7.18 (m, 12.1H, ArH), 7.21e7.26 (2xd,
3J ¼ 8.2 Hz, 2H, ArH, Z isomers), 7.34 (d, 3J ¼ 15.7 Hz, 0.5H,
CHCHCONH, EE isomer), 7.35 (d, 3J ¼ 15.7 Hz, 0.5H, CHCHCONH, EZ
4.1.3.1. General procedure for the preparation of homodimers with
different spacer length. To a solution of 12a or 12b (2.1 eq) in anh.
DMF (2 mL), PyBOP (2.2 eq) dissolved in anh. DCM (1 mL) was
added at 0 ꢀC under an argon atmosphere. The solution was stirred
for 5 min, then DIPEA (4.0 eq) was added dropwise, followed by an
aliquot of a freshly prepared stock solution of the respective
diamine (1 eq) in anh. DMF (0.1e1 mL). After 30 min on ice, the
mixture was allowed to warm to rt or was heated to 45 ꢀC. The
reaction was stopped after 20 he72 h depending on the used
diamine. Thereafter, the solvents were evaporated and the residue
isomer), 7.51e7.59 (m, 2.9H, ArH
þ
CHCHCONH). 13C NMR
(151 MHz, CD3OD): 13.83 E isomers, 13.89 Z isomers, 29.88 Z
d
isomers, 30.01 E isomers, 30.31e30.36, 38.03 EE isomer, 38.09 EZ
isomer, 38.11 EZ isomer, 38.17 ZZ isomer, 115.28 Z isomers, 116.03 E
isomers, 121.02 E isomers, 121.48 Z isomers, 127.22 Z isomers,
127.37 E isomers, 127.86 E isomers, 128.69 Z isomers, 128.94 Z iso-
mers, 129.01 E isomers, 130.84 Z isomers, 130.86 E isomers, 131.12 Z
isomers, 131.69 E isomers, 132.46 E isomers, 133.06 Z isomers,
133.54 E isomers, 134.62 Z isomers, 135.28 Z isomers, 135.59 E
isomers, 139.54 Z isomers, 139.68 E isomers, 141.53, 142.84 Z iso-
mers, 143.64 E isomers, 143.67 Z isomers, 143.91 E isomers, 146.80 E
isomers, 147.05 Z isomers, 156.68 Z isomers, 157.57 E isomers,
168.87. HRMS (m/z): calculated for C53H51N2O4 [MþH]þ: 777.3771,
found: 777.3742.
was dissolved in ethyl acetate and washed with 1
N HCl. The
aqueous layer was extracted twice with ethyl acetate. The com-
bined organic layers were washed with brine and dried over anh.
Na2SO4. Purification was achieved by two column chromatography
runs first with DCM and MeOH (95:5 / 93:7) then ethyl acetate
100% as eluent affording the respective homodiamides [30,54e56].
4.1.3.1.1. (E)-N,N’-(Ethane-1,2-diyl)bis[3-(4-((E/Z)-1-(4-
4.1.3.1.3. (E)-N,N’-(Butane-1,4-diyl)bis[3-(4-((E/Z)-1-(4-
hydroxyphenyl)-2-phenylbut-1-en-1-yl)phenyl)acrylamide]
(15a).
15a was synthesized according to the general procedure described
above: 78 mg of 12a (0.21 mmol), 121 mg of PyBOP (0.23 mmol),
0.07 mL of DIPEA (0.42 mmol) and 8.9 mg of 1,4-diaminobutane
(0.11 mmol) in 0.4 mL of anh. DMF were applied. The mixture
was stirred at rt for 24 h. 15a was obtained as yellowish, sparkling
powder (31 mg, 0.039 mmol, 37%). Purity: 97.4%. 1H NMR (700 MHz,
hydroxyphenyl)-2-phenylbut-1-en-1-yl)phenyl)acrylamide]
(13a).
13a was synthesized according to the general procedure described
above: 100 mg of 12a (0.27 mmol), 147 mg of PyBOP (0.28 mmol),
0.09 mL of DIPEA (0.51 mmol) and 7.7 mg of 1,2-diaminoethane
(0.13 mmol) in 0.3 mL of anh. DMF. The solution was stirred at
45 ꢀC for three days. 13a was obtained as a yellowish, sparkling
powder (29 mg, 0.040 mmol, 28%). Purity: 98.1%. 1H NMR
CD3OD, EE:EZ:ZZ ¼ 29:49:22):
d 0.92 (3xt, 6H, 3x CH2CH3),
1.57e1.59 (m, 1.2H, NHCH2CH2CH2CH2NH, EE isomer), 1.61e1.63 (m,

12
A. Knox et al. / European Journal of Medicinal Chemistry 192 (2020) 112191
2H, NHCH2CH2CH2CH2NH, EZ isomer), 1.65e1.67 (m, 0.9H,
NHCH2CH2CH2CH2NH, ZZ isomer), 2.47 (q, 3J ¼ 7.4 Hz, 1.8H, CH2CH3,
Z isomers), 2.52 (q, 3J ¼ 7.4 Hz, 2.2H, CH2CH3, E isomers), 3.29 (t,
3J ¼ 5.3 Hz 1.2H, NHCH2CH2CH2CH2NH, EE isomer), 3.33 (t,
was stirred at rt for 24 h. Upon extraction, the organic phase was
concentrated and the resulting precipitate filtered off and washed
with MeOH and DCM. 13b remained as a white powder (18 mg,
0.026 mmol, 32%). Purity: 95.4%. 1H NMR (400 MHz, DMSO‑d6):
3J
¼
6.8 Hz, 2H, NHCH2CH2CH2CH2NH, EZ isomer), 3.36 (t,
d 1.42e1.64 (m, 12H, CH2), 2.07e2.25 (m, 8H, CH2), 3.26e3.30 (m,
3J ¼ 6.0 Hz, 0.9H, NHCH2CH2CH2CH2NH, ZZ isomer), 6.41e6.45 (m,
4H, CH2), 6.56 (d, 3J ¼ 15.8 Hz, 2H, CHCHCONH), 6.68 (d, 3J ¼ 8.2 Hz,
4H, ArH), 6.86 (d, 3J ¼ 8.2 Hz, 4H, ArH), 7.08 (d, 3J ¼ 7.9 Hz, 4H, ArH),
7.39 (d, 3J ¼ 15.7 Hz, 2H, CHCHCONH), 7.47 (d, 3J ¼ 7.9 Hz, 4H, ArH),
8.21 (br, 2H, NH), 9.34 (br, 2H, OH). 13C NMR (100 MHz, DMSO‑d6):
2.9H, ArH, Z isomers þ CHCHCONH, E isomers), 6.59 (d, 3J ¼ 15.8 Hz,
0.5H, CHCHCONH, EZ isomer), 6.61 (d, 3J
¼
15.8 Hz, 0.4H,
CHCHCONH, ZZ isomer), 6.66 (d, 3J ¼ 8.8 Hz, 1.9H, ArH, Z isomers),
6.78 (2xd, 2.1H, ArH, E isomers), 6.83e6.92 (2xd, 3J ¼ 8.3 Hz, 2.1H,
ArH, E isomers), 7.03 (d, 3J ¼ 8.7 Hz, 2.1H, ArH, E isomers), 7.07e7.19
(m, 12.1H, ArH), 7.21e7.27 (2xd, 3J ¼ 8.2 Hz, 1.9H, ArH, Z isomers),
7.34 (d, 3J ¼ 15.7 Hz, 0.6H, CHCHCONH, EE isomer), 7.35 (d,
3J ¼ 15.8 Hz, 0.5H, CHCHCONH, EZ isomer), 7.49e7.57 (m, 2.8H,
ArH þ CHCHCONH, Z isomers). 13C NMR (176 MHz, CD3OD):
d
26.22, 28.16, 31.91, 31.95, 114.88, 121.59, 127.24, 129.96, 130.52,
132.67, 132.85, 133.55, 138.20, 138.48, 144.35, 155.85, 165.28. HRMS
(m/z): calculated for
693.3666.
C
46H49N2O4 [MþH]þ: 693.3687, found:
4.1.3.2.2. (E)-N,N’-(Propane-1,3-diyl)bis[3-(4-(cyclohexylidene(4-
hydroxyphenyl)methyl)phenyl)acrylamide] (14b). 14b was synthe-
sized according to the general procedure described above: 50 mg of
12b (0.15 mmol) in 0.5 mL of anh. DMF, 83 mg of PyBOP
(0.16 mmol), 0.05 mL of DIPEA (4 eq, 0.29 mmol) and 5.3 mg of 1,3-
diaminopropane (0.072 mmol) in 0.3 mL of anh. DMF. The mixture
was stirred for 24 h. After extraction and column chromatography
purification with DCM and MeOH (98:2 / 95:5) followed by
recrystallization from warm MeOH, 14b was obtained as a white
powder (34 mg, 0.048 mmol, 66%). Purity: 95.0%. 1H NMR
d
13.82 E isomers, 13.87 Z isomers, 27.84 EE isomer, 27.89 EZ isomer,
27.93 ZZ isomer, 29.86 Z isomers, 30.00 E isomers, 40.16 EE isomer,
40.18 EZ isomer, 40.22 EZ isomer, 40.24 ZZ isomer, 115.28 Z isomers,
116.03 E isomers, 121.10 E isomers, 121.56 Z isomers, 127.22 Z iso-
mers, 127.37 E isomers, 127.84 E isomers, 128.66 Z isomers, 128.93 Z
isomers, 129.00 E isomers, 130.84 Z isomers, 130.86 E isomers,
131.11 Z isomers, 131.68 E isomers, 132.45 E isomers, 133.05 Z iso-
mers, 133.58 E isomers, 134.66 Z isomers, 135.29 Z isomers, 135.61 E
isomers, 139.55 Z isomers, 139.69 E isomers, 141.39 E isomers,
141.40 Z isomers, 142.84 Z isomers, 143.64 E isomers, 143.68 Z iso-
mers, 143.90 E isomers, 146.75 E isomers, 147.01 Z isomers, 156.67 Z
isomers, 157.57 E isomers, 168.76 ZZ isomer, 168.78 EZ isomer,
168.80 EE isomer. HRMS (m/z): calculated for C54H51N2O4 [M ꢁ H]-:
791.3927, found: 791.3893.
(400 MHz, DMSO‑d6):
d
1.41e1.61 (m, 12H, CH2), 1.65 (p, 3J ¼ 6.5 Hz,
2H, NHCH2CH2CH2NH), 2.08e2.25 (m, 8H, CH2), 3.21 (q, 3J ¼ 6.6 Hz,
4H, NHCH2CH2CH2NH), 6.57 (d, 3J ¼ 15.8 Hz, 2H, CHCHCONH), 6.68
(d, 3J ¼ 8.5 Hz, 4H, ArH), 6.86 (d, 3J ¼ 8.5 Hz, 4H, ArH), 7.08 (d,
3J ¼ 8.0 Hz, 4H, ArH), 7.38 (d, 3J ¼ 15.7 Hz, 2H, CHCHCONH), 7.47 (d,
3J ¼ 8.0 Hz, 4H, ArH), 8.12 (t, 3J ¼ 5.1 Hz, 2H, NH), 9.35 (br, 2H, OH).
4.1.3.1.4. (E)-N,N’-(Pentane-1,5-diyl)bis[3-(4-((E/Z)-1-(4-
13C NMR (100 MHz, DMSO‑d6):
d 26.19, 28.13, 29.30, 31.89, 31.93,
hydroxyphenyl)-2-phenylbut-1-en-1-yl)phenyl)acrylamide]
(16a).
36.65, 114.85, 121.67, 127.19, 129.92, 130.50, 132.71, 132.81, 133.54,
16a was synthesized according to the general procedure described
above: 50 mg of 12a (0.14 mmol), 74 mg of PyBOP (0.23 mmol),
0.044 mL of DIPEA (0.26 mmol) and 6.6 mg of 1,5-diaminopentane
(0.065 mmol) in 0.7 mL of anh. DMF. The mixture was stirred at rt
for 48 h. 16a was obtained as a yellowish, sparkling powder (17 mg,
0.021 mmol, 33.0%). Purity: 95.7%. 1H NMR (600 MHz, CD3OD,
138.14, 138.27, 144.28, 155.83, 165.01. HRMS (m/z): calculated for
C
47H51N2O4 [MþH]þ: 707.3843, found: 707.3903.
4.1.3.2.3. (E)-N,N’-(Butane-1,4-diyl)bis[3-(4-(cyclohexylidene(4-
hydroxyphenyl)methyl)phenyl)acrylamide] (15b). 15b was synthe-
sized according to the general procedure described above: 58 mg of
12b (0.18 mmol) in 0.5 mL of anh. DMF, 96 mg of PyBOP
(0.18 mmol), 0.06 mL of DIPEA (4 eq, 0.33 mmol) and 7.4 mg of 1,4-
diaminobutane (0.084 mmol) in 0.5 mL of anh. DMF were stirred for
24 h. The next day, 15b was filtered off by suction, washed with
DCM and MeOH and remained as a white powder (45 mg,
0.064 mmol, 75%). Purity: 95.0%. 1H NMR (400 MHz, DMSO‑d6):
EE:EZ:ZZ ¼ 29:49:22):
d 0.89e0.95 (3xt, 6H, 3x CH2CH3), 1.36e1.49
(m, 2H, NHCH2CH2CH2CH2CH2NH), 1.53e1.67 (2xp, 4H,
2xNHCH2CH2CH2CH2CH2NH), 2.47 (q, 3J ¼ 7.4 Hz, 1.9H, CH2CH3, Z
isomers), 2.52 (2xq, 3J ¼ 7.4 Hz, 2.1H, 2xCH2CH3, E isomers),
3.25e3.35 (m, 4H, NHCH2CH2CH2CH2CH2NH), 6.41e6.45 (m, 2.9H,
ArH, Z isomers þ CHCHCONH, E isomers), 6.60 (d, 3J ¼ 15.8 Hz, 0.5H,
CHCHCONH, EZ isomer), 6.62 (d, 3J ¼ 15.8 Hz, 0.4H, CHCHCONH, ZZ
isomer), 6.66 (2xd, 3J ¼ 8.7 Hz, 1.9H, ArH, Z isomers), 6.78 (2xd,
3J ¼ 8.5 Hz 2.1H, ArH, E isomers), 6.86 (d, 3J ¼ 8.3 Hz, 2.1H, ArH, E
isomers), 7.03 (2xd, 3J ¼ 8.6 Hz, 2.1H, ArH, E isomers), 7.06e7.19 (m,
12.1H, ArH), 7.23 (d, 3J ¼ 8.2 Hz, 1.9H, ArH, Z isomers), 7.34 (d,
3J ¼ 15.7 Hz, 0.6H, CHCHCONH, EE isomer), 7.35 (d, 3J ¼ 15.7 Hz,
d
1.37e1.49 (m, 4H, NHCH2CH2CH2CH2NH),1.49e1.71 (m,12H, CH2),
2.01e2.82 (m, 8H, CH2), 3.12e3.25 (m, 4H, NHCH2CH2CH2CH2NH),
6.56 (d, 3J ¼ 15.8 Hz, 2H, CHCHCONH), 6.68 (d, 3J ¼ 8.2 Hz, 4H, ArH),
6.86 (d, 3J ¼ 8.1 Hz, 4H, ArH), 7.08 (d, 3J ¼ 7.8 Hz, 4H, ArH), 7.37 (d,
3J ¼ 15.7 Hz, 2H, CHCHCONH), 7.46 (d, 3J ¼ 7.8 Hz, 4H, ArH), 8.08 (t,
3J ¼ 4.9 Hz, 2H, NH), 9.32 (s, 2H, OH). 13C NMR (100 MHz, DMSO‑d6):
d
26.18, 26.72, 28.12, 31.87, 31.92, 38.39, 114.84, 121.76, 127.14,
0.5H,
CHCHCONH,
EZ
isomer),
7.50e7.57
(m,
2.8H,
13.84,
129.90, 130.47, 132.73, 132.81, 133.54, 138.13, 144.22, 155.81, 164.88.
HRMS (m/z): calculated for C48H53N2O4 [MþH]þ: 721.4000, found:
721.4090.
ArH þ CHCHCONH, Z isomers). 13C NMR (151 MHz, CD3OD):
d
13.90, 25.23, 29.89, 30.02, 30.04, 40.32, 40.38, 115.29, 116.04, 121.13,
121.60, 127.22, 127.37, 127.84, 128.66, 128.94, 129.01, 130.85, 130.87,
131.13, 131.69, 132.47, 133.07, 133.58, 134.67, 135.27, 135.58, 139.55,
139.69, 141.34, 142.83, 143.64, 143.68, 143.89, 146.74, 147.00, 156.70,
157.60, 168.77. HRMS (m/z): calculated for C53H51N2O4 [M ꢁ H]-:
805.4084, found: 805.4050.
4.1.3.2.4. (E)-N,N’-(Pentane-1,5-diyl)bis[3-(4-(cyclohexylidene(4-
hydroxyphenyl)methyl)phenyl)acrylamide] (16b). 16b was synthe-
sized according to the general procedure described above: 100 mg
of 12b (0.30 mmol) in 1 mL of anh. DMF, 165 mg of PyBOP
(0.32 mmol), 0.10 mL of DIPEA (0.58 mmol) and 15 mg of 1,5-
diaminopentane (0.08 mmol) in 0.7 mL of anh. DMF. The mixture
was stirred for 24 h at rt. After extraction and concentration of the
organic phase, the remaining solid was resuspended in DCM and
filtered by vacuum suction. After washing carefully with DCM and
cold MeOH, 16b was isolated as a white powder (59 mg, 0.08 mmol,
4.1.3.2. Cyclofenil-derived homodimers
4.1.3.2.1. (E)-N,N’-(Ethane-1,2-diyl)bis[3-(4-(cyclohexylidene(4-
hydroxyphenyl)methyl)phenyl)acrylamide] (13b). 13b was synthe-
sized according to the general procedure described above: 56 mg of
12b (0.17 mmol) in 0.5 mL of anh. DMF, 93 mg of PyBOP
(0.18 mmol), 0.056 mL of DIPEA (0.32 mmol) and 4.9 mg of 1,2-
diaminoethane (0.081 mmol) in 0.2 mL of anh. DMF. The mixture
56.2%), Purity: 95.2%. 1H NMR (400 MHz, DMSO‑d6):
d 1.29e1.39 (m,
2H, NHCH2CH2CH2CH2CH2NH), 1.44e1.61 (m, 16H, CH2), 2.17 (bm,
8H, CH2), 3.16 (bq, 3J ¼ 5.9 Hz, 4H, NHCH2CH2CH2CH2CH2NH), 6.57

A. Knox et al. / European Journal of Medicinal Chemistry 192 (2020) 112191
13
(d, 3J ¼ 15.8 Hz, 2H, CHCHCONH), 6.69 (d, 3J ¼ 8.4 Hz, 4H, ArH), 6.86
(d, 3J ¼ 8.4 Hz, 4H, ArH), 7.07 (d, 3J ¼ 8.0 Hz, 4H, ArH), 7.37 (d,
3J ¼ 15.7 Hz, 2H, CHCHCONH), 7.45 (d, 3J ¼ 8.0 Hz, 4H, ArH), 8.07 (bt,
3J ¼ 5.3 Hz, 2H, NH), 9.33 (s, 2H, OH). 13C NMR (101 MHz, DMSO‑d6):
manufacturer’s instructions to investigate the binding affinity to
the LBD. The recombinant LBD of ER /ER (4.2 nM), tagged with
glutathione S-transferase (GST) was mixed with a terbium labeled
anti-GST antibody (2 nM), Fluormone™ ES2 Green (3 nM) and 10
a
b
mL
d
24.11, 26.46, 28.44, 28.95, 31.19, 32.23, 115.20, 121.87, 127.56,
of a serial diluted stock solution of the compounds. Binding studies
were performed in a concentration-dependent manner. TR-FRET
was measured with an Enspire multimodal plate reader (Perki-
nElmer Life Sciences, Waltham, USA) using an excitation filter at
340/310 nm and emission filters for terbium at 495 nm and fluo-
rescein at 520 nm. The TR-FRET ratio was calculated by dividing the
emission signal of fluorescein by the emission signal of terbium.
The recruitment of coactivators was performed analogously
with the LanthaScreen®TR-FRET ER alpha Coactivator Assay (Invi-
trogen, Carlsbad, USA). For the antagonistic mode the assay was
performed with E2 4 nM.
130.22,130.81,132.94,133.36, 133.25,138.74,138.63,144.64,156.00,
165.58. HRMS (m/z): calculated for C49H55N2O4 [MþH]þ: 735.4156,
found: 735.4150.
4.1.3.2.5. (E)-N-[2-(Boc-amino)ethyl]-3-[4-((E/Z)-1-(4-
hydroxyphenyl)-2-phenylbut-1-enyl)phenyl]acrylamide (17). 17 was
synthesized according to the general procedure described above:
170 mg of 12a (1.0 eq, 0.41 mmol), 213 mg of PyBOP (1.0 eq,
0.41 mmol), 0.28 mL of DIPEA (4.0 eq, 1.62 mmol) and 72 mg of N-
Boc-1,2-diaminoethane (1.1 eq, 0.45 mmol) in 0.5 mL of anh. DMF.
The solution was stirred for 24 h at rt. 17 was obtained as a white-
yellow powder (105 mg, 0.20 mmol, 51%) [39,55e57]. 1H NMR
(200 MHz, CD3OD, E:Z ¼ 55:45):
d
0.93 (t, 3J ¼ 7.6 Hz, 3H, CH2CH3),
4.2.3. Luciferase reporter gene assay
€
1.42 (s, 5H, OC(CH3)3, E isomer), 1.44 (s, 4H, OC(CH3)3, Z isomer),
2.42e2.58 (m, 2H, CH2), 3.18e3.24 (m, 2H, CH2), 3.35e3.42 (m, 2H,
CH2), 6.39e6.56 (m, 2H, CHCHCONH þ ArH), 6.66 (d, 3J ¼ 8.2 Hz,
0.9H, ArH, Z isomer), 6.78 (d, 3J ¼ 8.4 Hz, 1.1H, ArH, E isomer), 6.88
(d, 3J ¼ 8.2 Hz, 1.1H, ArH, E isomer), 7.04 (d, 3J ¼ 8.6 Hz, 1.1H, ArH, E
isomer), 7.12e7.27 (m, 6.9H, ArH), 7.36 (d, 3J ¼ 15.6 Hz, 0.6H,
CHCHCONH, E isomer), 7.52e7.59 (m, 1.3H, CHCHCONH þ ArH, Z
isomers).
The transient transfection (TansIT-LT1, MoBiTec, Gottingen,
Germany) and the dual-luciferase reporter assay (Promega, Madi-
son, USA) were performed according to the manufacturer’s pro-
tocols. U2OS cells were seeded in 96-well plates (1 ꢃ 104 cells per
well) using McCoy’s 5A medium supplemented with 10% charcoal
dextran treated FBS as well as 1% penicillin/streptomycin and
incubated at 37 ꢀC in a humidified atmosphere (5% CO2/95% air) for
24 h. Then, the cells were transiently transfected with pSG5-ER
a
(1 ng) or pSG5-ER
b
(1 ng), respectively, p(ERE)2-lucþ (50 ng) and
4.1.3.3. (E)-N-(2-Aminoethyl)-3-[4-((E/Z)-1-(4-hydroxyphenyl)-2-
phenylbut-1-enyl)phenyl]acrylamide trifluoroacetate salt (18).
18 was prepared using 105 mg of 17 (0.20 mmol) in 1.0 mL anh.
DCM and 0.3 mL of TFA. The mixture was stirred under an argon
atmosphere at 0 ꢀC for 2 h, followed by the evaporation of the
solvent under reduced pressure. The residue was treated with
MeOH and DCM several times to remove remaining TFA and then
evaporated to dryness yielding a brownish oil (107 mg, 0.20 mmol,
pRenilla-CMV (0.5 ng) in phosphate-buffered saline (PBS) using
TransIT®-LT1. After 6e8 h, the compounds were added in a
concentration-dependent manner and incubated for 21 h, lucif-
erase activity was measured employing an Enspire multimodal
plate reader (PerkinElmer Life Sciences, Waltham, USA). Renilla
luciferase activity was used as internal control and for
normalization.
quant.) [57]. 1H NMR (200 MHz, CD3OD, E:Z ¼ 60:40):
d
0.92 (t,
4.2.4. Cellular uptake
3J ¼ 7.4 Hz, 3H, CH2CH3), 2.41e2.57 (m, 2H, CH2CH3), 3.06e3.16 (m,
2H, CH2), 3.51e3.62 (m, 2H, CH2), 6.40e6.68 (m, 3H,
The cellular uptake was quantified by fluorimetry on an Enspire
multimode plate reader and correlated to the protein content. MCF-
7 cells (0.25 ꢃ 106 cells per well) or COS-7 (0.32 ꢃ 106 cells per well)
were seeded (2 mL) in 6-well microtiter plates and kept at 37 ꢀC in a
humidified atmosphere (5% CO2/95% air) for 24 h followed by
further 24 h of drug incubation. The cells were rinsed with 2 mL of
3
CHCHCONH þ ArH), 6.79 (d, J ¼ 8.6 Hz, 1.2H, ArH, E isomer), 6.89
3
(d, J ¼ 8.2 Hz, 1.2H, ArH, E isomer), 7.02e7.28 (m, 8.8H, ArH, NH),
7.42 (d, 3J ¼ 16.0 Hz, 0.6H, CHCHCONH, E isomer), 7.55e7.65 (m,
1.2H, CHCHCONH þ ArH, Z isomer).
PBS and detached by adding 200
BioSciences, Pasching, Austria). Subsequently, the cells were har-
vested in 800 L of PBS and the cell suspension was centrifuged (rt,
8000 rcf, 3 min). The supernatant was discarded, and the isolated
mL of accutase (GE Healthcare
4.2. In vitro assays
m
4.2.1. General
The human osteosarcoma cell line U2OS, the human breast
cancer cell lines MCF-7, MDA-MB-231, and SKBr-3 as well as the
African green monkey kidney cell line COS-7 were obtained from
the cell line service (CLS, Eppelheim, Germany). The cells were
maintained as monolayer cultures. McCoy’s 5A medium supple-
mented with 10% fetal bovine serum (FBS) (both from Biochrome
GmbH, Berlin, Germany) was used for the U2OS and SKBr-3 cell
lines and Dulbecco’s modified eagle medium (DMEM) without
phenol red, with glucose (4.5 g Lꢁ1) (GE Healthcare, Pasching,
Austria), supplemented with 10% FBS and 1% pyruvate (GE
Healthcare) for MCF-7, MDA-MB-231, and COS-7 cell lines. They
were cultivated in a humidified atmosphere (5% CO2/95% air) at
37 ꢀC and passaged twice a week. DMSO was used as a solvent for
the investigated compounds. The final concentration of DMSO
never exceeded 0.1% in cell based assays. Vehicle treated controls
were always included.
cell pellets were washed with 1000 mL of PBS, resuspended,
centrifuged and then stored at ꢁ20 ꢀC for a maximum of two weeks
until further analysis. After thawing, the cell pellets were resus-
pended in 300
mL of distilled water and lysed by sonification
(setting parameter: 20 s, 9 cycles, 80e85% power). An aliquot was
used for the Bradford protein assay to relate the amount of drug
(nmol) to the protein content of the cell pellet (mg). The assay was
performed according to a previously described method [58]. For
fluorescence analysis, 100 mL of the lysates were diluted 1:1 with a
mixture of distilled water and MeOH (9:1) in a black 96-well plate
in duplicates. The excitation wavelength was set to 330 nm and the
emission was measured at 463 nm on an Enspire multimodal plate
reader (PerkinElmer Life Sciences, Waltham, USA). The average
emission of duplicates was calculated. The values represent the
means SD of ꢂ3 independent experiments.
4.2.5. Western blot
4.2.2. Binding assays
LanthaScreen®TR-FRET ER alpha/beta Competitive Binding As-
says (Invitrogen, Carlsbad, USA) were used according to the
MCF-7 cells (0.5 ꢃ 106 cells per well) were seeded in 6-well
plates in DMEM supplemented with 10% charcoal dextran treated
FBS and 1% pyruvate. For adhesion, the cells were incubated for 24 h

14
A. Knox et al. / European Journal of Medicinal Chemistry 192 (2020) 112191
overnight and then treated with 1
mM of compound dilutions for
CRediT authorship contribution statement
another 24 h. MG-132 (1 mM) was added half an hour before the
compounds and it was incubated for 4 h. After treatment, cells were
harvested and samples were lysed using a modified radio immu-
noprecipitation assay buffer (containing: 50 mM of Tris (pH ¼ 8.0),
150 mM of NaCl, 0.5% NP-40, 50 mM of NaF, 1 mM of Na3PO4, 1 mM
of phenylmethylsulfonyl fluoride (all from Sigma-Aldrich, Austria)
and protease inhibitors (EDTA-free; Roche, Austria)). Total protein
Alexandra Knox: Conceptualization, Data curation, Formal
analysis, Investigation, Methodology, Writing - original draft,
Writing - review & editing. Christina Kalchschmid: Data curation,
Formal analysis, Investigation, Methodology, Writing - original
draft, Writing - review & editing. Daniela Schuster: Resources,
Software, Investigation. Francesca Gaggia: Investigation. Claudia
Manzl: Resources, Investigation. Daniel Baecker: Investigation,
Writing - review & editing. Ronald Gust: Conceptualization, Su-
pervision, Writing - review & editing.
(30
(see above), then the proteins were processed by SDS-PAGE and
transferred onto nitrocellulose membrane (Amersham, GE
Healthcare, Austria). Membranes were probed with ER antibody
mg) concentration was determined by using the Bradford assay
a
a
(SP1, 1:1000, Invitrogen) and an HRP-labeled goat anti-rabbit
(DAKO, Agilent, Austria) was used as a secondary reagent. Anti-
Acknowledgements
body specific
b-actin (D6A8, 1:1000, Cell Signaling, Austria)
We thank Christoph Kreutz (Institute of Organic Chemistry,
University of Innsbruck) and Peter Schneider (Institute of Phar-
macy, Department of Pharmacognosy, University of Innsbruck) for
confirmed equal loading of proteins. Detection was assessed by
enhanced chemiluminescence (ECL, Thermo Scientific, Austria).
recording the 1H and 13C NMR spectra and Andre Betz (Institute of
ꢀ
4.2.6. In-Cell Western immunoassay
Pharmacy, Department of Pharmaceutical Chemistry, University of
Innsbruck) for assistance in the crystal violet assay. We also thank
Inte:Ligand GmbH for providing an academic license of LigandSc-
out. The Austrian Research Promotion Agency FFG [West Austrian
BioNMR 858017] is also kindly acknowledged.
Further investigations of the degradation were carried out using
an In-Cell Western™ Assay Kit and the CellTag™ 700 Stain (LI-COR,
Lincoln, USA). MCF-7 cells were seeded in 96-well plates
(1 ꢃ 104 cells per well) in DMEM supplemented with 10% charcoal
dextran treated FBS and 1% pyruvate. After 24 h, compounds were
added and incubated for another 24 h at 37 ꢀC in a humidified at-
mosphere (5% CO2/95% air). Medium was aspirated, cells were fixed
with a 3.7% formaldehyde solution and the assay was performed
Appendix B. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112191.
according to the manufacturer’s instructions. ER
a antibody (SP1,
1:250, Invitrogen) was used as primary antibody. Fluorescence in-
tensity was recorded and quantified using the Odyssey Infrared
Imaging System (LI-COR). DMSO and fulvestrant were used,
respectively, to set the basis for maximum response and maximum
Appendix A. Supplementary data
Supplementary data associated with this article is available free
of charge in the online version.
efficacy of ERa downregulation.
4.2.7. Crystal violet assay
References
The antiproliferative and cytotoxicity evaluation was performed
with the ER-positive MCF-7 cell line and the fibroblast-like cells
COS-7 according to a modified protocol previously described [58].
Cells were seeded in 96-well microtiter plates (2 ꢃ 103 cells per
well) in DMEM supplemented with 10% FBS and pyruvate. 24 h after
seeding, the complete medium with the compounds was added in
quadruples. After an incubation time of 72 h in a humidified at-
mosphere (5% CO2/95% air) at 37 ꢀC, the medium was aspirated,
cells were washed with PBS (GE Healthcare) and fixed with a so-
lution of 1% (v/v) glutaric dialdehyde in PBS. Cell biomass was
determined via staining of the chromatin of adherent cells with
crystal violet, extraction of the stain with ethanol (70% v/v) and
subsequent measurement of the absorbance at 590 nm. Cell
viability is expressed as percentage of cell viability of vehicle-
treated control which was set at 100%. Results are the
means SD of ꢂ3 independent experiments.
[1] V.C. Jordan, Antiestrogens and selective estrogen receptor modulators as
multifunctional medicines. 1. Receptor interactions, J. Med. Chem. 46 (2003)
883e908.
[2] V.C. Jordan, Antiestrogens and selective estrogen receptor modulators as
multifunctional medicines. 2. Clinical considerations and new agents, J. Med.
Chem. 46 (2003) 1081e1111.
[3] A.L. Rodriguez, A. Tamrazi, M.L. Collins, J.A. Katzenellenbogen, Design, syn-
thesis, and in vitro biological evaluation of small molecule inhibitors of es-
trogen receptor alpha coactivator binding, J. Med. Chem. 47 (2004) 600e611.
[4] A. Sun, T.W. Moore, J.R. Gunther, M.S. Kim, E. Rhoden, Y. Du, H. Fu, J.P. Snyder,
J.A. Katzenellenbogen, Discovering small-molecule estrogen receptor alpha/
coactivator binding inhibitors: high-throughput screening, ligand develop-
ment, and models for enhanced potency, ChemMedChem 6 (2011) 654e666.
[5] W. Qin, M. Xie, X. Qin, Q. Fang, F. Yin, Z. Li, Recent advances in peptidomi-
metics antagonists targeting estrogen receptor alpha-coactivator interaction
in cancer therapy, Bioorg. Med. Chem. Lett. 28 (2018) 2827e2836.
[6] K.E. Bergmann, C.H. Wooge, K.E. Carlson, B.S. Katzenellenbogen,
J.A. Katzenellenbogen, Bivalent ligands as probes of estrogen receptor action,
J. Steroid Biochem. Mol. Biol. 49 (1994) 139e152.
[7] A.L. LaFrate, K.E. Carlson, J.A. Katzenellenbogen, Steroidal bivalent ligands for
the estrogen receptor: design, synthesis, characterization and binding affin-
ities, Bioorg. Med. Chem. 17 (2009) 3528e3535.
[8] A.E. Wendlandt, S.M. Yelton, D. Lou, D.S. Watt, D.J. Noonan, Synthesis and
functional analysis of novel bivalent estrogens, Steroids 75 (2010) 825e833.
[9] M. Shan, A. Bujotzek, F. Abendroth, A. Wellner, R. Gust, O. Seitz, M. Weber,
R. Haag, Conformational analysis of bivalent estrogen receptor ligands: from
intramolecular to intermolecular binding, ChemBioChem 12 (2011)
2587e2598.
[10] M. Shan, K.E. Carlson, A. Bujotzek, A. Wellner, R. Gust, M. Weber,
J.A. Katzenellenbogen, R. Haag, Nonsteroidal bivalent estrogen ligands: an
application of the bivalent concept to the estrogen receptor, ACS Chem. Biol. 8
(2013) 707e715.
[11] Y. Wang, N.Y. Chirgadze, S.L. Briggs, S. Khan, E.V. Jensen, T.P. Burris, A second
binding site for hydroxytamoxifen within the coactivator-binding groove of
estrogen receptor beta, Proc. Natl. Acad. Sci. U.S.A. 103 (2006) 9908e9911.
[12] D.J. Kojetin, T.P. Burris, E.V. Jensen, S.A. Khan, Implications of the binding of
tamoxifen to the coactivator recognition site of the estrogen receptor, Endocr.
Relat. Cancer 15 (2008) 851e870.
4.2.8. EZ4U assay
Metabolic activity was evaluated analogously to the anti-
proliferative potency as described above. After 72 h of incubation,
the metabolic activity was investigated employing a modified MTT
assay (EZ4U Kit, Biomedica, Vienna, Austria) according to the
manufacturer’s protocol. The final data represent the means SD of
ꢂ3 independent experiments.
Declaration of competing interest
The authors declare that they have no known competing
financial interest or personal relationships that could have
appeared to influence the work reported in this paper.

A. Knox et al. / European Journal of Medicinal Chemistry 192 (2020) 112191
15
[13] A.C.W. Pike, A.M. Brzozowski, J. Walton, R.E. Hubbard, A.G. Thorsell, Y.L. Li,
J.Å. Gustafsson, M. Carlquist, Structural insights into the mode of action of a
pure antiestrogen, Structure 9 (2001) 145e153.
[14] B.M. Wittmann, A. Sherk, D.P. McDonnell, Definition of functionally important
mechanistic differences among selective estrogen receptor down-regulators,
Canc. Res. 67 (2007) 9549e9560.
limitations to the interpretation of experiments characterizing the activity of
individual isomers, J. Steroid Biochem. 22 (1985) 589e596.
[33] C. Malet, P. Spritzer, C. Cumins, D. Guillaumin, P. Mauvais-Jarvis, F. Kuttenn,
Effect of 4-hydroxytamoxifen isomers on growth and ultrastructural aspects
of normal human breast epithelial (HBE) cells in culture, J. Steroid Biochem.
Mol. Biol. 82 (2002) 289e296.
[15] A. Lai, M. Kahraman, S. Govek, J. Nagasawa, C. Bonnefous, J. Julien, K. Douglas,
J. Sensintaffar, N. Lu, K.J. Lee, A. Aparicio, J. Kaufman, J. Qian, G. Shao,
R. Prudente, M.J. Moon, J.D. Joseph, B. Darimont, D. Brigham, K. Grillot,
R. Heyman, P.J. Rix, J.H. Hager, N.D. Smith, Identification of GDC-0810 (ARN-
810), an orally bioavailable selective estrogen receptor degrader (SERD) that
demonstrates robust activity in tamoxifen-resistant breast cancer xenografts,
J. Med. Chem. 58 (2015) 4888e4904.
[34] D.C. Hilt, V. Masini-Eteve, R. Fedynec, B. Taravella, Chemically stable compo-
sitions of 4-hydroxytamoxifen, 2005. WO 2005/092310.
[35] R.V. Weatherman, N.J. Clegg, T.S. Scanlan, Differential SERM activation of the
estrogen receptors (ER
[36] V.C. Jordan, M.M. Collins, L. Rowsby, G. Prestwich,
a
and ER
b
) at AP-1 sites, Chem. Biol. 8 (2001) 427e436.
monohydroxylated
A
metabolite of tamoxifen with potent antioestrogenic activity, J. Endocrinol. 75
(1977) 305e316.
[16] C. De Savi, R.H. Bradbury, A.A. Rabow, R.A. Norman, C. de Almeida,
D.M. Andrews, P. Ballard, D. Buttar, R.J. Callis, G.S. Currie, J.O. Curwen,
C.D. Davies, C.S. Donald, L.J.L. Feron, H. Gingell, S.C. Glossop, B.R. Hayter,
S. Hussain, G. Karoutchi, S.G. Lamont, P. MacFaul, T.A. Moss, S.E. Pearson,
M. Tonge, G.E. Walker, H.M. Weir, Z. Wilson, Optimization of a novel binding
motif to (E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-
2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (AZD9496), a
potent and orally bioavailable selective estrogen receptor downregulator and
antagonist, J. Med. Chem. 58 (2015) 8128e8140.
[37] K.E. Allen, E.R. Clark, V.C. Jordan, Evidence for the metabolic activation of non-
steroidal antioestrogens: a study of structure-activity relationships, Br. J.
Pharmacol. 71 (1980) 83e91.
[38] Z. Nawaz, D.M. Lonard, A.P. Dennis, C.L. Smith, B.W. O’Malley, Proteasome-
dependent degradation of the human estrogen receptor, Proc. Natl. Acad. Sci.
U.S.A. 96 (1999) 1858e1862.
[39] I. Laios, F. Journe, D. Nonclercq, D.S. Vidal, R.A. Toillon, G. Laurent, G. Leclercq,
Role of the proteasome in the regulation of estrogen receptor alpha turnover
and function in MCF-7 breast carcinoma cells, J. Steroid Biochem. Mol. Biol. 94
(2005) 347e359.
[17] G.S. Tria, T. Abrams, J. Baird, H.E. Burks, B. Firestone, L.A. Gaither, L.G. Hamann,
G. He, C.A. Kirby, S. Kim, F. Lombardo, K.J. Macchi, D.P. McDonnell, Y. Mishina,
J.D. Norris, J. Nunez, C. Springer, Y. Sun, N.M. Thomsen, C. Wang, J. Wang,
€
€
[40] H. Reile, H. Birnbock, G. Bernhardt, T. Spruß, H. Schonenberger, Computerized
determination of growth kinetic curves and doubling times from cells in
microculture, Anal. Biochem. 187 (1990) 262e267.
B. Yu, C.L. Tiong-Yip, S. Peukert, Discovery of LSZ102,
a potent, orally
bioavailable selective estrogen receptor degrader (SERD) for the treatment of
estrogen receptor positive breast cancer, J. Med. Chem. 61 (2018) 2837e2864.
[18] Y.L. Wu, X. Yang, Z. Ren, D.P. McDonnell, J.D. Norris, T.M. Willson, G.L. Greene,
Structural basis for an unexpected mode of SERM-mediated ER antagonism,
Mol. Cell 18 (2005) 413e424.
[19] T.M. Willson, B.R. Henke, T.M. Momtahen, P.S. Charifson, K.W. Batchelor,
D.B. Lubahn, L.B. Moore, B.B. Oliver, H.R. Sauls, J.A. Triantafillou, S.G. Wolfe,
P.G. Baer, 3-[4-(1,2-Diphenylbut-1-enyl)phenyl]acrylic acid: a non-steroidal
estrogen with functional selectivity for bone over uterus in rats, J. Med.
Chem. 37 (1994) 1550e1552.
[41] J. Liu, S. Zheng, S. Guo, C. Zhang, Q. Zhong, Q. Zhang, P. Ma, E.V. Skripnikova,
M.R. Bratton, T.E. Wiese, G. Wang, Rational design of a boron-modified tri-
phenylethylene (GLL398) as an oral selective estrogen receptor down-
regulator, ACS Med. Chem. Lett. 8 (2017) 102e106.
[42] I.N. White, F. de Matteis, A. Davies, L.L. Smith, C. Crofton-Sleigh, S. Venitt,
A. Hewer, D.H. Phillips, Genotoxic potential of tamoxifen and analogues in
female Fischer F344/n rats, DBA/2 and C57BL/6 mice and in human MCL-5
cells, Carcinogenesis 13 (1992) 2197e2203.
[43] K. Niwa, S. Morishita, M. Hashimoto, T. Itoh, J. Fujimoto, H. Mori, T. Tamaya,
Effects of tamoxifen on endometrial carcinogenesis in mice, Jpn. J. Cancer Res.
89 (1998) 502e509.
[44] B. Abderrahman, V.C. Jordan, Improving long-term adjuvant anti-oestrogenic
therapy for breast cancer, Clin. Pharm. 8 (2016), https://doi.org/10.1211/
CP.2016.20201203.
[20] D. Bentrem, R. Dardes, H. Liu, J. MacGregor-Schafer, J. Zapf, V.C. Jordan, Mo-
lecular mechanism of action at estrogen receptor alpha of a new clinically
relevant antiestrogen (GW7604) related to tamoxifen, J. Endocrinol. 142
(2001) 838e846.
[21] S.W. Fanning, L. Hodges-Gallagher, D.C. Myles, R. Sun, C.E. Fowler, I.N. Plant,
B.D. Green, C.L. Harmon, G.L. Greene, P.J. Kushner, Specific stereochemistry of
OP-1074 disrupts estrogen receptor alpha helix 12 and confers pure anti-
estrogenic activity, Nat. Commun. 9 (2018) 2368.
[45] B. Abderrahman, V.C. Jordan, Successful targeted therapies for breast cancer:
the worcester foundation and future opportunities in women’s health,
Endocrinology 159 (2018) 2980e2990.
[46] S.E. Fawell, R. White, S. Hoare, M. Sydenham, M. Page, M.G. Parker, Inhibition
of estrogen receptor-DNA binding by the “pure” antiestrogen ICI 164,384
appears to be mediated by impaired receptor dimerization, Proc. Natl. Acad.
Sci. U.S.A. 87 (1990) 6883e6887.
[47] S. Dauvois, R. White, M.G. Parker, The antiestrogen ICI 182780 disrupts es-
trogen receptor nucleocytoplasmic shuttling, J. Cell Sci. 106 (1993)
1377e1388.
[22] K.J.
Kieser,
D.W.
Kim,
K.E.
Carlson,
B.S.
Katzenellenbogen,
J.A. Katzenellenbogen, Characterization of the pharmacophore properties of
novel selective estrogen receptor downregulators (SERDs), J. Med. Chem. 53
(2010) 3320e3329.
[23] J. Min, V.S. Guillen, A. Sharma, Y. Zhao, Y. Ziegler, P. Gong, C.G. Mayne,
S. Srinivasan, S.H. Kim, K.E. Carlson, K.W. Nettles, B.S. Katzenellenbogen,
J.A. Katzenellenbogen, Adamantyl antiestrogens with novel side chains reveal
a spectrum of activities in suppressing estrogen receptor mediated activities
in breast cancer cells, J. Med. Chem. 60 (2017) 6321e6336.
[48] PerkinElmer Informatics ®. 1998-2014.
[49] G. Jones, P. Willett, R.C. Glen, A.R. Leach, R. Taylor, Development and valida-
tion of a genetic algorithm for flexible docking, J. Mol. Biol. 267 (1997)
727e748.
[24] I. Paterni, C. Granchi, J.A. Katzenellenbogen, F. Minutolo, Estrogen receptors
alpha (ER
Steroids 90 (2014) 13e29.
a
) and beta (ER
b
): subtype-selective ligands and clinical potential,
[50] G. Wolber, T. Langer, LigandScout: 3-D pharmacophores derived from
protein-bound ligands and their use as virtual screening filters, J. Chem. Inf.
Model. 45 (2005) 160e169.
[25] M.J. Li, H.M. Greenblatt, O. Dym, S. Albeck, A. Pais, C. Gunanathan, D. Milstein,
H. Degani, J.L. Sussman, Structure of estradiol metal chelate and estrogen
receptor complex: the basis for designing a new class of selective estrogen
receptor modulators, J. Med. Chem. 54 (2011) 3575e3580.
[26] J.F. Eaddy, D. Heyer, S.R. Katamreddy, M.T. Martin, M.S. McClure,
A.S. Randhawa, V. Samano, J.A. Ray, Preparation of acylox-
ydiphenylbutenylcinnamates as estrogen receptor modulator prodrugs, 2005.
WO2005/033056A2.
[51] A.K. Shiau, D. Barstad, P.M. Loria, L. Cheng, P.J. Kushner, D.A. Agard,
G.L. Greene, The structural basis of estrogen receptor/coactivator recognition
and the antagonism of this interaction by tamoxifen, Cell 95 (1998) 927e937.
€
[52] S. Mocklinghoff, R. Rose, M. Carraz, A. Visser, C. Ottmann, L. Brunsveld, Syn-
thesis and crystal structure of a phosphorylated estrogen receptor ligand
binding domain, ChemBioChem 11 (2010) 2251e2254.
€
[53] L.L.C. Schrodinger, The PyMOL Molecular Graphics System, 2006. Version 0.99.
[27] J.F. Eaddy, D. Heyer, S.R. Katamreddy, M.T. Martin, M.S. MCClure,
A.S. Randhawa, V. Samano, J.A. Ray, Triphenylethylene compounds as selec-
tive estrogen receptor modulators, 2008. EP1692127A2.
[28] M. Fan, E.L. Rickert, L. Chen, S.A. Aftab, K.P. Nephew, R.V. Weatherman,
Characterization of molecular and structural determinants of selective es-
trogen receptor downregulators, Breast Cancer Res. Treat. 103 (2007) 37e44.
[29] V. Lubczyk, H. Bachmann, R. Gust, Investigations on estrogen receptor binding.
The estrogenic, antiestrogenic, and cytotoxic properties of C2-alkyl-
substituted 1,1-bis(4-hydroxyphenyl)-2-phenylethenes, J. Med. Chem. 45
(2002) 5358e5364.
[54] L.H. Lin, L.W. Lee, S.Y. Sheu, P.Y. Lin, Study on the stevioside analogues of
steviolbioside, steviol, and isosteviol 19-alkyl amide dimers: synthesis and
cytotoxic and antibacterial activity, Chem. Pharm. Bull. 52 (2004) 1117e1122.
[55] E. Rivero-Buceta, P. Carrero, E.G. Doyagüez, A. Madrona, E. Quesada,
ꢀ
ꢀ
M.J. Camarasa, M.J. Perez-Perez, P. Leyssen, J. Paeshuyse, J. Balzarini, J. Neyts,
ꢀ
A. San-Felix, Linear and branched alkyl-esters and amides of gallic acid and
other (mono-, di- and tri-) hydroxy benzoyl derivatives as promising anti-
HCV inhibitors, Eur. J. Med. Chem. 92 (2015) 656e671.
[56] M. Shan, Design, synthesis, and evaluation of bivalent estrogen ligands, Freie
€
Universitat Berlin, Berlin, 2011. PhD Thesis.
[30] J. Coste, D. Le-Nguyen, B. Castro, PyBOP®: a new peptide coupling reagent
devoid of toxic by-product, Tetrahedron Lett. 31 (1990) 205e208.
[31] V.W. Winkler, M.A. Nyman, R.S. Egan, Diethylstilbestrol cis-trans isomeriza-
tion and estrogen activity of diethylstilbestrol isomers, Steroids 17 (1971)
197e207.
[57] C.B. Vu, J.E. Bemis, M.R. Jirousek, J.C. Milne, J.J. Smith, Preparation of fatty acid
acylated salicylates for the treatment of inflammatory disorders, 2010.
US2010/0184730A1.
[58] V. Obermoser, D. Baecker, C. Schuster, V. Braun, B. Kircher, R. Gust, Chlorinated
cobalt alkyne complexes derived from acetylsalicylic acid as new specific
antitumor agents, Dalton Trans. 47 (2018) 4341e4351.
[32] J.A. Katzenellenbogen, K.E. Carlson, B.S. Katzenellenbogen, Facile geometric
isomerization of phenolic non-steroidal estrogens and antiestrogens: